Human C3b/C4b receptor (CR1)

ABSTRACT

The present invention relates to the C3b/C4b receptor (CR1) gene and its encoded protein. The invention also relates to CR1 nucleic acid sequences and fragments thereof comprising 70 nucleotides and their encoded peptides or proteins comprising 24 amino acids. The invention further provides for the expression of the CR1 protein and fragments thereof. The genes and proteins of the invention have uses in diagnosis and therapy of disorders involving complement activity, and various immune system or inflammatory disorders. In specific embodiments of the present invention detailed in the examples sections infra, the cloning, nucleotide sequence, and deduced amino acid sequence of a full-length CR1 cDNA and fragments thereof are described. The expression of the CR1 protein and fragments thereof is also described. Also described is the expression of a secreted CR1 molecule lacking a transmembrane region. The secreted CR1 molecule is shown to be useful in reducing damage caused by inflammation and in reducing myocardial infarct size and preventing reperfusion injury.

This application is a division of U.S. application Ser. No. 08/350,238,filed Dec. 6, 1994 (now abandoned), which is a continuation of U.S.application Ser. No. 08/026,134, filed Feb. 24, 1993 (now abandoned),which is a division of U.S. application Ser. No. 07/332,865, filed04/03/89 (now U.S. Pat. No. 5,212,071), which is a continuation-in-partof U.S. application Ser. No. 07/176,532, filed Apr. 1, 1988 (nowabandoned).

Pursuant to the provisions of 35 U.S.C. §202(c), it is herebyacknowledged that the Government has certain rights in this invention,which was made in part with funds from the National Institutes ofHealth.

TABLE OF CONTENTS

Page  1. Introduction 8  2. Background of the Invention 8 2.1. TheComplement System 8 2.2. The C3b/C4b Complement Receptor (CR1) 9 2.3.Abnormalities of CR1 in Human Disease 12  3. Summary of the Invention 143.1. Definitions 15  4. Description of the Figures 15  5. DetailedDescription of the Invention 23 5.1. Isolation of the CR1 Gene 24 5.2.Expression of the Cloned CR1 Gene 30 5.3. Identification andPurification of the 34 Expressed Gene Product 5.4. Structure of the CR1Gene and Protein 36 5.4.1. Genetic Analysis 36 5.4.2. Protein Analysis37 5.5. CR1-Related Derivatives, Analogues, and 39 Peptides 5.6. Uses ofCR1 40 5.6.1. Assays and Diagnosis 40 5.6.2. Therapy 42  6. Example: TheCloning and Sequencing of the Human 45 C3b/C4b Receptor (CR1) 6.1.Materials and Methods 46 6.1.1. Isolation and Sequence of CR1 Tryptic 46Peptides 6.1.2. Isolation of cDNA Clones and Genomic 46 Clones 6.1.3.DNA Sequence Analysis 47 6.2. Results 48 6.2.1. Nucleotide Sequence ofthe CR1 Gene 48 6.2.2. Analysis of the Nucleotide and Amino 49 AcidSequence of CR1 6.3. Discussion 54  7. Example: CR1 5′ cDNA SequencesContain a Fourth 59 Long Homologous Repeat 7.1. Materials and Methods 597.1.1. Construction of a cDNA Library 59 7.1.2. Isolation of Clones,Probes, and 60 DNA Sequence Analysis 7.2. Results 60 7.3. Discussion 63 8. Example: Expression of Recombinant Human CR1 64 8.1. Construction ofpBSABCD Containing the 65 Entire CR1 Coding Sequence 8.2. Constructionand Assay of Plasmid piABCD, 68 a Mammalian Expression Vector Containingthe Entire CR1 Coding Sequence 8.3. Expression of CR1 Fragments 718.3.1. Construction of Deletion Mutants 71 piBCD, piABD, piACD, piAD,piBD, piCD and piD 8.3.2. Construction of Deletion Mutants piP1, 74piE1, piE2, piE-2, piU1, piU-2 and piA/D  9. Example: Identification ofC3b and C4b Binding 76 Domains 9.1. Assays and Results 76 9.2.Discussion 79 10. Example: Demonstration of Factor I Cofactor 81Activity 11. Example: Expression of Recombinant Soluble CR1 83 11.1.Materials and Methods 84 11.1.1. Enzyme Digestions 84 11.1.2. DNAFragment Isolations 84 11.1.3. Transfection into Mammalian Cells 8511.1.4. CHO Transfectant Cell Culture 85 11.1.5. ELISA for the Detectionof 86 CR1 Levels 11.1.5.1. CR1 Standards 86 11.1.5.2. CR1 ELISA 86 11.2.Genetic Modifications of CR1 Coding Sequences 87 11.2.1. Construction ofpBSCR1c 88 11.2.2. Construction of pBSCR1s 89 11.2.3. Construction ofpBM-CR1c 89 11.2.4. Construction of Deletion Mutants 90 pT-CR1c1,pT-CR1c2, pT-CR1c3, pT-CR1c4, and pT-CR1c5 11.2.4.1. pT-CR1c1 9011.2.4.2. pT-CR1c2 91 11.2.4.3. pT-CR1c3 91 11.2.4.4. pT-CR1c4 9211.2.4.5. pT-CR1c5 92 11.3. Expression of Soluble CR1 93 11.3.1.Construction of pTCS Series of 93 Expression Vectors 11.3.1.1.Construction of pEAXgpt 94 11.3.1.2. Construction of pMLEgpt 9611.3.1.3. Construction of pTCSgpt 96 11.3.1.4. Construction of pTCSdhfr97 11.3.1.5. Construction of pTCSneo 97 11.3.2. Expression and Assay ofPlasmids 97 pBSCR1c, pBSCR1s and pBM-CR1c, Mammalian Expression VectorsContaining Soluble CR1 Coding Sequences 11.3.2.1. Expression of CR1 98Constructs Truncated at Different Positions Within the CR1 cDNA11.3.2.2. Expression of sCR1c in 101 Two Different Expression Systems11.3.3. Expression and Assay of Plasmids 102 pT-CR1c1, pT-CR1c2,pT-CR1c3, pT-CR1c4, and pT-CR1c5, Mammalian Expression VectorsContaining Soluble CR1 Coding Sequences 12. Example: Production andPurification of Soluble CR1 103 12.1. Large Scale Production of SolubleCR1 104 12.1.1. Production of sCR1 in Serum-Free Media 105 12.1.2.Conclusions 107 12.2. Purification of Soluble CR1 107 12.2.1. AntibodyAffinity Column 108 Purification 12.2.1.1. Methods 108 12.2.1.2. Results108 12.2.2. CR1 Purification by HPLC 109 12.2.2.1. Methods 10912.2.2.1.1. Starting Material 12.2.2.1.2. Cation Exchange 109 HPLCProcedure 12.2.2.1.3. Anion Exchange 110 HPLC Procedure 12.2.2.1.4.Western Blot 110 Analysis 12.2.2.2. Results 110 12.2.2.3.Characterization of 111 Purified Soluble CR1 12.2.2.4. Conclusions 11213. Example: Demonstration of In Vitro Activity of 112 Soluble CR1 13.1.Inhibition of the Neutrophil Oxidative Burst 112 13.1.1. Materials andMethods 113 13.1.1.1. Materials 113 13.1.1.2. Preparation of 113Neutrophils 13.1.1.3. Preparation of Yeast 114 Particles 13.1.1.4.Activation of 114 Neutrophils by Purified C5a 13.1.1.5. Activation of114 Neutrophils by Purified C5a in Human Serum or Plasma 13.1.1.6.Activation of 114 Neutrophils by Yeast Particle-Activated Human Serum orPlasma 13.1.2. Results 115 13.1.2.1. C5a Induces an Oxygen 115 Burst inHuman Neutro- phils Which Can be Measured Using DCFDA 13.1.2.2. HumanSerum Blocks the 115 Oxygen Burst Effects of Purified C5a on Neutrophils13.1.2.3. Heparinized Plasma does 115 not Block the Effects of C5a onNeutrophils 13.1.2.4. sCR1 Present During 116 Complement ActivationBlocks C5a Generation 13.2. Inhibition of Complement Mediated Hemolysis116 13.2.1. Methods 116 13.2.2. Results 117 13.3. Inhibition of C3a andC5a Production 120 13.3.1. Methods 120 13.3.2. Results 121 14. Example:Demonstration of Functional In Vivo 121 Therapeutic Activity of SolubleCR1 14.1. Soluble CR1 Demonstrates In Vivo Function 121 in a ReversedPassive Arthus Reaction 14.1.1. Materials and Methods 122 14.1.2.Results 123 14.1.3. Effect of Intradermal Administration 123 of SolubleCR1 14.2. Pharmacokinetics of In Vivo Administered sCR1 124 14.3. sCR1Reduces Infarct Size in Rats with 126 Reperfused Infarcted Myocardium14.3.1. Methods 126 14.3.1.1. Induction of Rat 126 Myocardial Infarct14.3.1.2. Morphological Analysis of 127 Experimental Infarcts:Preparation of Hearts for Study 14.3.2. Results 128 14.3.3. Conclusions128 15. Deposit of Microorganisms 128

1. INTRODUCTION

The present invention relates to the C3b/C4b receptor (CR1) gene and itsencoded protein. The invention also relates to CR1 nucleic acidsequences and fragments thereof comprising 70 nucleotides, and theirencoded peptides or proteins comprising 24 amino acids. The inventionalso provides for the expression of the CR1 protein and fragmentsthereof. The CR1 nucleic acids and proteins have use in the diagnosis ortherapy of disorders involving complement activity, and variousinflammatory and immune disorders.

2. BACKGROUND OF THE INVENTION 2.1. The Complement System

The complement system is a group of proteins that constitutes about 10percent of the globulins in the normal serum of humans (Hood, L. E., etal., 1984, Immunology, 2d Ed., The Benjamin/Cummings Publishing Co.,Menlo Park, Calif., p. 339). Complement (C) plays an important role inthe mediation of immune and allergic reactions (Rapp, H. J. and Borsos,T, 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts(Meredith), New York). The activation of complement components leads tothe generation of a group of factors, including chemotactic peptidesthat mediate the inflammation associated with complement-dependentdiseases. The sequential activation of the complement cascade may occurvia the classical pathway involving antigen-antibody complexes, or by analternative pathway which involves the recognition of certain call wallpolysaccharides. The activities mediated by activated complementproteins include lysis of target calls, chemotaxis, opsonization,stimulation of vascular and other smooth muscle cells, and functionalaberrations such as dogranulation of mast cells, increased permeabilityof small blood vessels, directed migration of leukocytes, and activationof B lymphocytes and macrophages (Eisen, H. N., 1974, Immunology,Harper.& Row Publishers, Inc. Hagerstown, Md., p. 512).

During proteolytic cascade steps, biologically active peptide fragments,the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, 1977,WHO Tech. Rep. Ser. 606:5 and references cited therein), are releasedfrom the third (C3), fourth (C4), and fifth (C5) native complementcomponents (Hugli, T. E., 1981, CRC Crit. Rev. Immunol. 1:321; Bult, H.and Herman, A. G., 1983, Agents Actions 13:405).

2.2. The C3b/C4b Complement Receptor (CR1)

The human C3b/C4b receptor, termed CR1, is present on erythrocytes,monocytes/macrophages,. granulocytes, B cells, some T cells, splenicfollicular dendritic cells, and glomerular podocytes (Fearon, D. T.,1980, J. Exp. Med. 152:20, Wilson, J. G., at al., 1983, J. Immunol.131:684; Reynes, M., et al., 1985, J. Immunol. 135:2687; Gelfand, N. C.,et al., 1976, N. Engl. J. Med. 295:10; Kazatchkine, M. D., et al., 1982,Clin. Immunol. Immunopathol. 27:170). CR1 specifically binds C3b, C4b,and iC3b. A soluble form of the receptor has been found in plasma thathas ligand binding activity and the same molecular weight asmembrane-associated CR1 (Yoon, S. H. and Fearon, D. T., 1985, J.Immunol. 134:3332). CR1 binds C3b and C4b that have covalently attachedto immune complexes and other complement activators, and theconsequences of these interactions depend upon the call type bearing thereceptor (Fearon, D. T. and Wong, W. W., 1983, Ann. Rev. Immunol.1:243). Erythrocyte CR1 binds immune complexes for transport to theliver (Cornacoff, J. B., at al., 1983, J. Clin. Invest. 71:236; Nedof,N. E., at al., 1982, J. Exp. Med. 156:1739) CR1 on neutrophils andsonocytes internalizes bound complexes, either by adsorptive endocytosisthrough coated pits (Fearon, D. T., et al., 1981, J. Exp. Med. 153:1615;Abrahamson, D. R. and Fearon, D. T., 1983, Lab. Invest. 48:162) or byphagocytosis after activation of the receptor by phorbol esters,chemotactic peptides, or proteins that are present in the extracellularmatrix, such as fibronectin and laminin (Newman, S. L., et al., 1980, J.Immunol. 125:2236; Wright, S. D. and Silverstein, S. C., 1982, J. Exp.Med. 156:1149; Wright, S. D., et al., 1983, J. Exp. Med. 158:1338).Phosphorylation of CR1 may have a role in the acquisition of phagocyticactivity (Changelian, P. S. and Fearon, D. T., 1986, J. Exp. Med.163:101). The function of CR1 on B lymphocytes is less defined, althoughtreatment of these cells with antibody to CR1 enhanced their response tosuboptimal doses of pokeweed mitogen (Daha, M. R., et al., 1983,Immunobiol. 164:227 (Abstr.)). CR1 on follicular dendritic cells maysubserve an antigen presentation role. (Klaus, G.G.B., et al., 1980,Immunol. Rev. 53:3).

CR1 can also inhibit the classical and alternative pathway C3/C5convertases and act as a cofactor for the cleavage of C3b and C4b byfactor I, indicating that CR1 also has complement regulatory functionsin addition to serving as a receptor (Fearon, D. T., 1979, Proc. Natl.Acad. Sci. U.S.A. 76:5867; Iida, X. and Nussenzweig, V., 1981, J. Exp.Med. 153:1138). In the alternative pathway of complement activation, thebimolecular complex C3b,Bb is a C3 activating enzyme (convertase). CR1(and factor H, at higher concentrations) can bind to C3b and can alsopromote the dissociation of C3b,Bb. Furthermore, formation of C3b,CR1(and C3b,H) renders C3b susceptible to irreversible proteolyticinactivation by factor I, resulting in the formation of inactivated C3b(iC3b). In the classical pathway of complement activation, the complexC4b,2a is the C3 convertase. CR1 (and C4 binding protein, C4bp, athigher concentrations) can bind to C4b, and can also promote thedissociation of C4b,2a. The binding renders C4b susceptible toirreversible proteolytic inactivation by factor I through cleavage toC4c and C4d (inactivated complement proteins.)

CR1 is a glycoprotein composed of a single polypeptide chain. Fourallotypic forms of CR1 have been found, differing by increments of˜40,000-50,000 daltons molecular weight. The two most common forms, theF and S allotypes, also termed the A and B allotypes, have molecularweights of 250,000 and 290,000 daltons (Dykman, T. R., et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:1698; Wong, W. W., et al., 1983, J.Clin. Invest. 72:685), respectively, and two rarer forms have molecularweights of 210,000 and >290,000 daltons (Dykman, T. R., et al., 1984, J.Exp. Med. 159:691; Dykman, T. R., et al., 1985, J. Immunol. 134:1787).These differences apparently represent variations in the polypeptidechain of CR1, rather than glycosylation state, because they were notabolished by treatment of purified receptor protein with endoglycosidaseF (Wong, W. W., et al., 1983, J. Clin. Invest. 72:685), and they wereobserved when receptor allotypes were biosynthesized in the presence oftunicamycin (Lublin, D. M., et al., 1986, J. Biol. Chem., 261:5736). Allfour CR1 allotypes have C3b-binding activity (Dykman, T. R., et al.,1983, Proc. Natl. Acad. Sci. U.S.A. 80:1698; Wong, W. W., et al., 1983,J. Clin. Invest. 72:685; Dykman, T. R., et al., 1984, J. Exp. Ned.159:691; Dykman T. R., et al., 1985, J. Immunol. 134:1787).

Two nonoverlapping restriction fragments of a CR1 cDNA were shown tocrosshybridize under conditions of high, stringency (Wong, W. W., etal., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7711). Both cDNA probes alsohybridized to multiple restriction fragments of genomic DNA, most ofwhich were common to both probes (id.). The existence of repetitivecoding sequences within CR1 was confirmed by sequence comparisons(Klickstein, L. B., et al., 1985, Complement 2:44 (Abstr.)). Inaddition, the CR1 gene has been shown to have repetitive interveningsequences by the demonstration of crosshybridization of a genomic probelacking coding sequences to several genomic restriction fragments (Wong,W. W., at al., 1986, J. Exp. Med. 164:15:1). Further, DNA from anindividual having the larger S allotype had an additional restrictionfragment hybridizing to this genomic probe when compared with DNA froman individual having the F allotype, suggesting that duplication ofgenomic sequences occurred in association with the higher. molecularweight CR1 allele (id.).

CR1 has been shown to have homology to complement receptor type 2 (CR2)(Weis, J. J., et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:5639-5643).

2.3. Abnormalities of CR1 in Human Disease

Diminished expression of CR1 on erythrocytes of patients with systemiclupus erythematosus (SLE) has been reported by investigators fromseveral geographic regions, including Japan (Miyakawa et al., 1981,Lancet 2:493-497; Minota et al., 1984, Arthr. Rheum. 27:1329-1335), theUnited States (Iida et al., 1982, J. Exp. Med. 155:1427-1438; Wilson etal., 1982, N. Engl. J. Med. 307:981-986) and Europe (Walport et al.,1985, Clin. Exp. Immunol. 59:547; Jouvin et al., 1986, Complement3:88-96; Holme et al., 1986, Clin. Exp. Immunol. 63:41-48). Taken as agroup, patients have an average number of receptors per cell that is50-60% that of normal populations. An early report noted that CR1 numberon erythrocytes varied inversely with disease activity, with lowestnumbers occurring during periods of most severe manifestations of SLE,and higher numbers being observed during periods of remission in thesame patient (Iida et al., 1982, J. Exp. Ned. 155:1427-1438). C1 numberhas also been found to correlate inversely with serum levels of immunecomplexes, with serum levels of C3d, and with the amounts oferythrocyte-bound C3dg, perhaps reflecting uptake ofcomplement-activating immune complexes and deposition on the erythrocyteas an “innocent bystander” (Ross et al., 1985, J. Immunol.135:2005-2014; Holme et al., 1986, Clin. Exp. Immunol. 63:41-48; Walportet al., 1985, Clin. Exp. Immunol. 59:547). A patient with SLE lackingCR1 on erythrocytes was found to have an auto-antibody to CR1 (Wilson etal., 1985, J. Clin. Invest. 76:182-190). Decreased titers of theanti-CR1 antibody coincided with improvement of the patient's clinicalcondition and with partial reversal of the receptor abnormality.Anti-CR1 antibody has been detected in two other SLE patients (Cook etal., 1986, Clin. Immunol. Immunopathol. 38:135-138). Recently, acquiredloss of erythrocyte CR1 in the setting of active SLE and hemolyticanemia was demonstrated by observing the rapid loss of the receptor fromtransfused erythrocytes (Walport et al., 1987, Clin. Exp. Immunol.69:501-507).

The relative loss of CR1 from erythrocytes has also been observed inpatients with Human Immunodeficiency Virus (HIV) infections (Tausk, F.A., et al., 1986, J. Clin. Invest. 78:977-982) and with lepromatusleprosy (Tausk, F. A., et al., 1985, J. Invest. Dermat. 85:58s-61s).

Abnormalities of complement receptor expression in SLE are not limitedto erythrocyte CR1. Relative deficiencies of total cellular CR1 ofneutrophils and plasma membrane CR1 of B lymphocytes of the SLE patientshave bean shown to occur (Wilson at al., 1986, Arthr. Rheum.29:739-747).

In patients with Type IV SLE nephritis, all detectable CR1 antigen islost from podocytes, whereas in less severe forms of SLE nephritis andin non-SLE types of proliferative nephritis, includingmembranoproliferative glomerulonephritis Types I and II, CR1 expressionon glomerular podocytes does not differ from normal (Kazatchkine at al.,1982, J. Clin. Invest. 69:900-912; Emancipator et al., 1983, Clin.Immunol. Imunopathol. 27: 170-175). However, patients having Type IV SLEnephritis do not have fewer numbers of erythrocyte CR1 than do SLEpatients having other types of renal lupus or no nephritis (Jouvin etal., 1986, Complement 3:88-96).

In vivo complement activation up-regulates CR1 expression at the plasmamembrane of neutrophils (Lee, J., et al., 1984, Clin. Exp. Immunol.56:205-214; Moore, F. D., Jr., et al., 1986, N. Engl. J. Med.314:948-953).

3. SUMMARY OF THE INVENTION

The present invention relates to the C3b/C4b receptor (CR1) gene and itsencoded protein. The invention also relates to CR1 nucleic acidsequences and fragments thereof comprising 70 nucleotides and theirencoded pepticles or proteins comprising 24 amino acids. The inventionfurther provides for the expression of the CR1 protein and fragmentsthereof. The genes and proteins of the invention have uses in diagnosisand therapy of disorders involving complement activity, and variousimmune system or inflammatory disorders.

In specific embodiments of the present invention detailed in theexamples sections infra, the cloning, nucleotide sequence, and deducedamino acid sequence of a full-length CR1 cDNA and fragments thereof aredescribed. The expression of the CR1 protein and fragments thereof isalso described. Expression of the CRI protein and its fragments whichcontain binding sites for C3b and/or C4b, and which exhibit factor Icofactor activity, is obtained.

Also described in the examples infra are the production and purificationof soluble CR1 molecules, which molecules are shown to betherapeutically useful for the treatment of inflammatory reactions andin the reduction of myocardial infarct size and prevention ofreperfusion injury.

3.1. Definitions

Ad2 MLP=adenovirus 2 major late promoter

C=complement

C3(ma)=methylamine-treated C3

C4bp=C4 binding protein

CMV=cytomegalovirus

CR1=complement receptor type 1, the C3b/C4b receptor

CR2=complement receptor, type 2

DCFDA=dichlorofluorescin diacetate

HPLC=high performance liquid chromatography

iC3b=inactivated C3b

LHR=long homologous repeat

mAb=monoclonal antibody

PAGE=polyacrylamide gel electrophoresis

RPAR=reverse passive Arthrus reaction

SCR=short consensus repeat

sCR1=soluble CR1 molecule

4. DESCRIPTION OF THE FIGURES

FIG. 1 (A through P). Nucleotide and amino acid sequence of the entireCR1 coding region. The sequence begins with the first nucleotidefollowing the octamer EcoRI linker in clone. λT109.1. Nucleotide number1531 of this sequence is the first nucleotide 5′ of nucleotide number 1of the sequence depicted in FIGS. 3A-3D. The strand corresponding to themRNA is shown, with the deduced amino acid sequence presented below. Theputative signal sequence encoded by nucleotide numbers 28-147 isbracketed.

FIG. 2. Restriction map of 5.5 kb of human CR1 cDNA. The black barindicates the cDNA, restriction sites are H. HindIII; B, BamHI; R,EcoRI; P, PstI; A, ApaI; S, SacI; G. BglII; K, KpnI. The cDNA clonesfrom which the sequence was derived are shown below the map. The arrowsindicate the direction and extent of sequence analysis by thedideoxynucleotide chain termination method. cDNA clones were oriented onthe-basis of restriction Maps and overlapping sequence identity.

FIG. 3 (A through D). Nucleotide sequence of 5.5 kb of human CR1 cDNA.The strand corresponding to the mRNA is shown and base number 1(corresponding to base number 1532 of FIG. 1D is the first base afterthe EcoRI linker in the most 5′ clone. The stop codon is underlined. The110-bp sequence in the box was found between nucleotides 147 and 148(arrow) and is believed to represent a portion of an interveningsequence.

FIG. 4. Dot matrix analysis of the nucleotide sequence of 5.5 kb ofhuman CR1 cDNA. A dot was plotted if there was at least a 40 bp of 90 bpmatch. The dark line bisecting the square diagonally indicates theidentity of the sequence with itself. The two additional parallel darklines 1.35 and 2.7 kb from the line of identity represent two tandem,direct long homologous repeats (LHRs) of 1.35 kb each. The six lighter,dashed lines between two LHRs correspond to short consensus repeats of˜0.2 kb. The short consensus repeats (SCRs) extend 0.4 kb beyond thelong homologous repeats.

FIG. 5 (A through C). (A) A schematic diagram of the CR1 protein (TM)transmembrane region, (CYT) cytoplasmic region, (3′UT) 3′ untranslatedsequence. (B) Deduced amino acid sequence of human CR1. Each residue isshown in the one letter code (Lehninger, A. L., 1975, Biochemistry, 2dEd., Worth Publishers, Inc., New York, p. 72). The residues in the longhomologous repeats have been aligned to illustrate their homology. Allthe residues in LHR-B are shown, and a residue is given for LHR-C andLHR-D only where it is different from that in LHR-B. A hydropathyprofile is aligned under the COOH-terminus of the protein to illustratethe presumptive transmembrane region. A stretch of four positivelycharged residues immediately after the hydrophobic sequence isoverlined. The six amino acid sequence with 67% homology to the site ofprotein kinase c phosphorylation in the epidermal growth factor receptoris underlined.

FIG. 6 (A through B). (A) Alignment of the SCR. of CR1. The repeats arenumbered 1-23 from NH₂-terminal to COOH-termnal. Spaces have beenintroduced to maximize the alignment. The boxes represent invariantresidues and the vertical arrows indicate positions of amino acidconservation. A residue is deemed conserved if it, or a conservativesubstitution, is present in at least half of the SCRs. The horizontalarrow indicates an SCR that was also sequenced from CR1 genomic clone2.38 and is encoded by a single exon. (B) Restriction map, sequencingstrategy, and partial sequence of genomic clone λ2.38. The restrictionsites are: (B) BamHI, (S) SacI, (E) EcoRV, (K) KpnI, (P) PstI. Thehorizontal arrow indicates direction and extent of sequencing and thevertical arrows indicate the exon-intron boundaries.

FIG. 7. Alignment of the consensus sequence of the SCRs of proteinsknown to have this structure. Spaces were introduced to maximize thealignment. A residue is deemed conserved as in FIG. 5, except for thoseproteins having only one or two SCRs, in which a residue is conserved ifit is present in at least half of the other proteins. The dashescorrespond to nonconserved positions. The underlined portions of CR2 andC2b indicate that no sequence information has been published in thisregion for these proteins. The boxes indicate the invarianthalf-cystines. The number to the right of the sequence represents thenumb r of SCR used to generate the consensus sequence. The proteinabbreviations and references for the sequence data used to determine theconsensus sequences are:. (CR1) complement receptor type 1, (H) factor H(Kristensen, T., et al., 1986, J. Immunol. 136:3407), (C4bp) C4 bindingprotein (Chung, L. P., at al., 1985, Biochem. J. 230:133), (CR2)complement receptor type 2 (Weis, J. J., et al., 1986, Proc. Natl. Acad.Sci. U.S.A. 83:5639), (Ba) proteolytic fragment of factor B (Morley, B.J. and Campbell, R. D., 1984, EMBO J. 3:153), (C2b) proteolytic fragmentof C2 (Gagnon, J., 1984, Philos. Trans. R. Soc. Lond. B Biol. Sci.306:301), (C1r) r subunit of C1 (Leytus, S. P., et al., 1986,Biochemistry 25:4855), (XIIIb) b subunit of factor XIII (Ichinose, A.,et al., 1986, Biochemistry 25:4633), (β2GP1) β2 glycoprotein I (Lozier,J., et al., 1984, Proc. Natl., Acad. Sci. U.S.A. 81:3640), (Hap)haptoglobin (Kurosky, A., et al., 1980, Proc. Natl. Acad. Sci. U.S.A.77:3388), (IL-2-R) the interleukin-2 receptor (Leonard, W. J., et al.,1985, Science 230:633). Asterisk indicates that incomplete sequence isavailable.

FIG. 8 (A through B). (A) Schematic diagram of the proposed structure ofhuman CR1. The COOH-terminal cytoplasmic region is on the right side ofthe lipid bilayer. 30 SCRs are arrayed linearly on the extracellularside of the plasma membrane. The brackets indicate the LHRs (B). Theinset is aLn enlargement of a single SCR to illustrate the triple loopstructure.

FIG. 9. Restriction map of the insert of the plasmid, pBSABCD, encodinghuman CR1. Indicated within the box delineating the region containingthe coding sequence are the nine fragments of eight cDNA clones thatwere ligated to form the CR1 construct. The brackets designate thepositions of LHR-A, -B, -C, and -D, respectively. The lines below thebox represent the positions of the newly isolated 5° cDNA clones. Therestriction sites are: A, ApaI, B, BamHI; G, BglII; H, HindIII; K, KPnI;M, BspMII; P, PstI; R, EcoRI; and S, SacI.

FIG. 10. The deduced amino acid sequence of the 5′ cDNA clones encodingthe seven SCRs of LHR-A, and alignment of this sequence with thecorresponding SCRs of LHR-B, -C, and -D. The four cysteines that areconserved in each SCR are underlined. A residue is shown for LHR-B, -Cand -D only where it is different from that in LHR-A.

FIG. 11. Restriction maps of the expression plasmids, (B) piABCD and (A)pMTABCD. Pm_(MT) and P_(CMV) represent the murine metallothionein andcytomegalovirus immediate early promoters, respectively

FIG. 12 (A through D). Analysis by phase contrast (panels a and c) andimmunofluorescent (panels b and d) microscopy of COS cells transfectedwith piABCD (panels a and b) and CDM8 vector alone (panels c and d) ,respectively, and indirectly stained with YZ1 monoclonal anti-CRiantibody and fluorescein-labelled goat anti-mouse F(ab′)₂.

FIG. 13 (A through D). Analysis of C3b- and C4b-binding by COS cellsexpressing recombinant CR1. COS cells transfected with piABCD (panels aand c) or with the CDM8 vector alone (panels b and d) were incubatedwith EAC4b(lim),3b (panels; a and b) or with EAC4b (panels c and d) andexamined for formation of rosettes by phase contrast microscopy.

FIG. 14. Analysis of recombinant CR1 expressied by transfected COS cellsby SDS-PAGE. COS cells transfected with the CDM8 vector alone (lanes 1and 4) and with piABCD (lanes 2 and 5), respectively, and erythrocytesfrom an individual having the F and S allotypes of CR1 (lanes 3 and 6)were surface labelled with ¹²⁵I. Detergent lysates of the cells weresequentially immunoadsorbed with Sepharose-UPC10 (lanes 1-3) andSepharose-YZ1 (lanes 4-6) and the eluates analyzed by SDS-PAGE undernon-reducing conditions and autoradiography.

FIG. 15. Cleavage of ¹²⁵I-C3(ma) by factor I in the presence ofimmunomobilized recombinant CR1. Replicate samples of ¹²⁵I-C3(ma) weretreated with factor I in the presence of factor H (lane 1),Sepharose-UPC10 preincubated with the lysate of COS cells transfectedwith the CDM8 vector alone (lane 2), Sopharos*-UPC10 preincubated withthe lysate of piABCD-transfected COS cells (lane 3), Sepharose-YZ1preincubated with the lysate of CDM8-transfected COS cells (lane 4), and6 μl (lane 5), 12 μl (lane 6) and 25 μl (lane 7) of Sepharose-YZ1 thathad been preincubated with the lysate of piABCD-transfected COS cells.Samples of ¹²⁵I-labelled C3(ma) were also treated in the absence offactor I with 25 μl of Sepharose-YZ1 that had been preincubated with thelysate of piABCD-transfected COS cells (lane 8). After reduction, the¹²⁵I-C3(ma) was analyzed by SDS-PAGE and autoradiography.

FIG. 16. The cDNA constructs encoding the CR1 deletion mutants. Thepositions of the cDNA segments encoding the four LHRs are indicated bythe brackets above the full length piABCD construct on which are shownthe restriction sites used for preparation of the deletion mutants. ThecDNA restriction fragments remaining in each of the mutants areindicated by the solid lines. The restriction sites are: A, APaI; B,BsmI; E, BstEII; and P, PstI.

FIG. 17. Comparison of recombinant deletion mutants of CR1 with the wildtype F and S allotypes of CR1. Detergent lysates of ¹²⁵I-surfacelabelled erythrocytes (lanes 1 and 7) and COS cells transfected withCDM8 vector alone (lanes 2 and 8), piABCD (lanes 3 and 9), piBCD (lanes4 and 10), piCD (lanes 5 and 11) and piD (lanes 6 and 12), respectively,were immunoprecipitated with Sepharose-UPC10 anti-levan antibody (lanes1-6), Sepharose-YZ-1 anti-CR1 monoclonal antibody (lanes 7-11) andrabbit anti-CR1 antibody and Sepharose-protein A (lane 12),respectively. The eluates were subjected to SDS-PAGE under reducingconditions and autoradiography.

FIG. 18. Cleavage of ¹²⁵I-C3(ma) by factor I in the presence of COScells expressing full length and deletion mutants of CR1. Replicatesamples of ¹²⁵I-C3(ma) were incubated with COS cells transfected withthe CDM8 vector alone (lanes 1 and 7), piABCD (lanes 2 and 8), piAD(lanes 3 and 9), piBD (lanes 4 and 10), picD (lanes 5 and 11), and piD(lanes 6 and 12), respectively, in the absence (lanes 1-6) or presence(lanes 7-12) of factor I. Samples of ¹²⁵I-C3(ma) also were incubatedwith factor H and factor I (lane 13) and with factor I alone (lane 14),respectively. After reduction, the ¹²⁵I-C3(ma) was analyzed by SDS-PAGEand autoradiography.

FIG. 19. Schematic model depicting the types of SCRs comprising each LHRof CR1, and the predicted sites determining the specificities of thereceptor for C3b and C4b. The secondary binding specificities of theseare indicated by the parentheses.

FIG. 20. A schematic diagram illustrating the DNA regions remaining inthe soluble CR1 DNA constructs. The regions of the full length CR1 cDNAare indicated by the boxes along the top of the figure.

FIG. 21. A schematic diagram illustrating the major elements in the pTCSseries of expression vectors.

FIG. 22. A diagram of the expression vector pTCSgpt. The polyadenylationsite is from the murine Ig kappa sequences (NBRF Nucleic databaseaccession #Kcms, bp 1306-1714); the Ad2 MLP and tripartite regions arefrom the Ad2 sequence (NBRF Nucleic database accession #Gdad2, bp5791-6069); the SV40 early promoter is from the SV40 genone (NBRFNucleic Database accession #GSV40W). The gpt gene, ampicillin gene andbacterial origin of replication are from the vector pSV2gpt (ATCCAccession No. 37145).

FIG. 23. 4-20% SDS-PAGE of antibody affinity purified sCR1. Non-reducing(lanes 1, 2, 3) and reducing (lanes 4, 5, 6) conditions. Lanes 1, 3:molecular weight markers; lanes 3, 5: cell culture supernatant startingmaterial; lanes 4, 6: sCR1 purified by antibody affinity chromatography.

FIG. 24. Cation exchange HPLC elution profile. Eluted protein wasmonitored by absorbance at 280 nm (y-axis). The absorbance of both theflow-through (0-100 minutes) and the eluted sCR1 (150-165 minutes) wereboth offscale. The x-axis represents the elution time in minutes.

FIG. 25. 4-20% gradient SDS-PAGE of cation and anion exchange HPLCpurified sCR1. SDS-polyacryamide gels were run under non-reducingconditions. Lane 1, an aliquot of bioreactor supernatant; lane 2, analiquot of bioreactor supernatant dialyzed against cation HPLC startingbuffer; lane 3, an aliquot of the eluted sCR1 peak from a cationexchange HPLC column; lane 4, an aliquot of the sCR1 peak from thecation exchange HPLC column dialyzed into starting buffer for anionHPLC; lanes 5 and 6, aliquots of two different fractions of eluted sCR1from anion HPLC.

FIG. 26 (A through G). C5a induction of an oxygen burst in humanneutrophils. Following a C5a induced oxygen burst, DCFDA became oxidizedand brightly fluoresced. Fluorescent intensity, as determined by flowcytometry, is measured on the x-axis and number of cells on the y-axis.Panel a, profile and gate for the cells; panel b, 0 minutes after C5aaddition; panel c, 1 minute; panel d, 2 minutes; panel e, 3 minutes;panel f, 4 minutes; panel g, 20 minutes. This DCFDA assay gives asensitive indication of C5a.

FIG. 27 (A through C). Activation of human complement in the presence ofsCR1 shows reduced C5a activity in the DCFDA assay. Panel a,unstimulated cells; panel b, control without sCR1 showing a high degreeof fluorescence; panel c, DCFDA assay in the presence of sCR1 showing areduction of 75% in fluorescent intensity. y-axis is number of cells andx-axis is fluorescent intensity.

FIG. 28. Inhibition of classical pathway C5a and C3a production in humanserum by sCR1. Similar profiles were observed for either antibodyaffinity purified or HPLC purified sCR1.

FIG. 29. Inhibition of complement-mediated hemolysis by recombinantsCR1. Similar profiles were observed for antibody affinity purified orHPLC purified sCR1.

FIG. 30 (A through B). Gross morphology of RPAR in sCR1-treated (left)and untreated (right) rats. (A) Both rats received an intravenousinjection of ovalbumin, followed by an intradermal injection of amixture of either sCR1 (left rat) or PBS (right rat) withanti-ovalbumin, neat (left site); anti-ovalbumin, ½ dilution (middlesite) or rabbit IgG (right site). The injections were performed induplicate; top and bottom rows gave identical results. The rat whichreceived sCR1 had barely visible changes, while the untreated ratdeveloped full symptoms of RPAR. (B) The dermal surface of the skinbiopsies from (A). The biopsy from the untreated rat (right) developedclearly visible lesion, while the biopsy from the sCR1-treated rat(left) showed normal morphology.

FIG. 31 (A through B). Light microscopy of skin biopsies fromsCR1-treated (A) and untreated (B) rats.

(A) Perivascular accumulation of polymorphonuclear and mononuclear cellswas observed, however, no extensive infiltration of neutrophils orextravasation of erythrocytes was seen. (B) Extensive infiltration ofpolymorphonuclear cells and extravasation of erythrocytes wasidentified.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the C3b/C4b receptor (CR1) gene andits encoded protein. The invention is also directed to CR1 nucleic acidsequences and fragments thereof comprising 70 nuclootidis and theirencoded peptides or proteins comprising 24 amino acids. The inventionfurther provides for the expression of the CR1 protein and fragmentsthereof. Such CR1 sequences and proteins have value in diagnosis andtherapy of inflammatory or immune system disorders, and disordersinvolving complement activity.

In a specific embodiment, the invention relates to soluble CR1 moleculesand the expression, purification, and uses thereof. As used herein, theterm “soluble CR1 molecules” shall mean portions of the CR1 proteinwhich, in contrast to the native CR1 proteins, are not expressed on thecell surface as membrane proteins. As a particular example, CR1molecules which substantially lack a. transmembrane region are solubleCR1 molecules. In a preferred embodiment, the soluble CR1 molecules aresecreted by a cell in which they are expressed.

In specific embodiments of the present invention detailed in theexamples sections infra, the cloning and complete nucleotide and deducedamino acid sequence of the full-length CR1 cDNA, and of fragmentsthereof, and the expression of the encoded CR1 products, are described.The expression of CR1 and fragments thereof, with binding sites for C3band/or C4b, and which inhibit factor I cofactor activity, is alsodescribed. The invention is further illustrated by the production andpurification of soluble, truncated CR1 molecules. In specific examples,such molecules are demonstrated to be therapeutically useful in reducinginflammation, and in reducing myocardial infarct size and preventingreperfusion injury.

5.1. Isolation of the CR1 Gene

The complete coding sequence of the CR1 gene and its deduced amino acidsequence is presented in FIG. 1.

Any human cell can potentially serve as the nucleic acid source for themolecular cloning of the CR1 gene. Isolation of the CR1 gene involvesthe isolation of those DNA sequences which encode a protein displayingCR1-associated structure or properties, e.g., binding of C3b or C4b orimmune complexes, modulating phagocytosis, immune stimulation orproliferation, and regulation of complement. The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired human cell.(See, for example, Maniatis et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K., Vol. I, II.) Cells which can serve as sourcesof nucleic acid for cDNA cloning of the CR1 gene include but are notlimited to monocytes/macrophages, granulocytes, B cells, T cells,splenic follicular dendritic cells, and. glomerular podocytes. Clonesderived from genomic DNA may contain regulatory and intron DNA regionsin addition to coding regions; clones derived from cDNA will containonly, exon sequences. Whatever the source, the CR1 gene should bemolecularly cloned into a suitable vector for propagation of. the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired CR1 gene. The DNA maybe cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the CR1 gene may be accomplished in a number ofways. For example, if an amount of a CR1 gene or its specific RNA, or afragment thereof, is available and can be purified and labeled,, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton, W. and Davis, R., 1977, Science 196:180;Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A.72:3961). Those DNA fragments with substantial homology to the probewill hybridize. If a purified CR1-specific probe is unavailable, nucleicacid fractions enriched in CR1 may be used as a probe, as an initialselection procedure. As an example, the probe representing B cell cDNAfrom which messages expressed by fibroblasts have been subtracted can beused. It is also possible to identify the appropriate fragment byrestriction enzyme digestion(s) and comparison of fragment sizes withthose expected according to a known restriction map if such isavailable. Further selection on the basis of the properties of the gene,or the physical, chemical, or immunological properties of its expressedproduct, as described infra, can be employed after the initialselection.

The CR1 gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary iRNAs by hybridization. SuchDNA fragments may represent available, purified CR1 DNA, or DNA that hasbeen enriched for CR1 sequences. Immunoprecipitation analysis orfunctional assays (e.g., for C3b or C4b binding, or promotion ofphagocytosis or immune stimulation, or complement regulation, etc.) ofthe in vitro translation products of the isolated mRNAs identifies themRNA and, therefore, the complementary DNA fragments that contain theCR1 sequences. In addition, specific mRNAs may be selected by adsorptionof polysomes isolated from cells to immobilized antibodies specificallydirected against. CR1. A radiolabeled CR1 cDNA can be synthesized usingthe selected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identify theCR1 DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the CR1 genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the CR1 gene.For example, as described supra, RNA for cDNA cloning of the CR1 genecan be isolated from cells including but not limited tomonocytes/macrophages, granulocytes, B cells, T cells, dendritic cells,and podocytes. In a preferred embodiment, tonsilar cells can serve asthe source of mRNA for cDNA cloning (See Section 6.1.2, infra). Othermethods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid or CDM8 plasmid (Seed, B., 1987, Nature329:840-842) or derivatives. Recombinant molecules can be introducedinto host cells via transformation, transfection, infection,electroporation, etc.

In an alternative method, the CR1 gene may be identified and isolatedafter insertion into a suitable cloning vector, in a “shot gun”approach. Enrichment for the CR1 gene, for example, by sizefractionation, can be done before insertion into the cloning vector.

The CR1 gene is inserted into a cloning vector which can be used totransform, transfect, or infect appropriate host cells so that manycopies of the gene sequences are generated. In a specific embodiment,the cloning vector can be the CDM8 vector, which can be used to achieveexpression in a mammalian host cell. The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and CR1 gene maybe modified by homopolymeric tailing.

Identification of the cloned CR1 gene can be accomplished in a number ofways based on the properties of the DNA itself, or alternatively, on thephysical, immunological, or functional properties of its encodedprotein. For example, the DNA itself may be detected by plaque or colonynucleic acid hybridization to labeled probes (Benton, W. and Davis, R.,1977, Science 196:180; Grunstein, M. and Hoghess, D., 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961). Alternatively, the presence of the CR1 genemay be detected by assays based on properties of its expressed product.For example, cDNA clones, or DNA clones which hybrid-select the propermRNAs, can be selected which produce a protein that, e.g., has similaror identical electrophoretic migration, isoelectric focusing behavior,proteolytic digestion maps, C3b and/or C4b and/or immune complex bindingactivity, complement regulatory activity, effects on phagocytosis orimmune stimulation, or antigenic properties as known for CR1. Using anantibody to CR1, the CR1 protein may be identified by binding of labeledantibody to the putatively CR1-synthesizing clones, in an ELISA(enzyme-linked immunosorbent assay)-type procedure.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated CR1 gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

In a particular embodiment, CR1 cDNA clones in a CDM8 vector can betransfected into COS (monkey kidney) cells for large-scale expressionunder the control of the cytomegalovirus promoter (see Section 8,infra).

If the ultimate goal is to insert the gene into virus expressionvectors, such as vaccinia virus or adenovirus, the recombinant DNAmolecule that incorporates the CR1 gene can be modified so that the geneis flanked by virus sequences that allow for genetic recombination incells infected with the virus so that the gene can be inserted into theviral genome.

After the CR1 DNA-containing clone has been identified, grown, andharvested, its DNA insert may be characterized as described in Section5.4.1, infra.

When the genetic structure of the CR1 gene is known, it is possible tomanipulate the structure for optimal use in the present invention. Forexample, promoter DNA may be ligated 5′ of the CR1-coding sequence, inaddition to or replacement of the native promoter to provide forincreased expression of the protein. Expression vectors which expressCR1 deletion mutants can also be made, to provide for expression ofdefined fragments of the CR1 sequence (see the example sections, infra).In a particular embodiment, deletion mutants can be constructed whichencode fragments of the CR1 protein that exhibit the desired C3b and/orC4b binding activity (see Section 9, infra), e.g., LHR-A for binding ofC4b, or LHR-C for binding of C3b. In a preferred embodiment, anexpression vector which encodes a CR1 molecule with a deletion of thetransmembrane region can be used to produce a soluble CR1 molecule (seethe examples sections 11-14, infra). Many manipulations are possible,and within the scope of the present invention.

5.2. Expression of the Cloned CR1 Gene

The nucleotide sequence coding for the CR1 protein (FIG. 1) or a portionthereof, can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The necessarytranscriptional and translation signals can also be supplied by thenative CR1 gene and/or its flanking regions. A variety of host-vectorsystems may be utilized, to express the protein-coding sequence. Thoseinclude but are not limited to mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect call systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophageDNA, plasmid DNA or cosmid DNA. The expression elements of these vectorsvary in their strength and specificities. Depending on the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements may be used. For instance, when cloning inmammalian cell systems, promoters isolated from the genome of mammaliancells or from viruses that grow in these cells (e.g., adenovirus, simianvirus 40, cytomegalovirus) may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the inserted sequences.

Specific initiation signals are also required for efficient translationof inserted protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where the entire CR1gene including its own initiation codon and adjacent sequences areinserted into the appropriate expression vectors, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the CR1 coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon, mustbe provided. The initiation codon must furthermore be in phase with thereading frame of the protein coding sequences to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene. consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).

In a specific embodiment, a soluble CR1 molecule can b expressed. Such asoluble molecule can be produced by use of recombinant DNA techniques todelete the DNA sequences encoding the CR1 transmembrane region (seeSections 11-14, infra). As demonstrated infra, the ability to express asoluble CR1 molecule is not limited to any one genetic modification ofthe CR1 nucleic acid sequence; as long as the nucleic acid sequenceencoding a substantial portion of the CR1 transmembrane region isdeleted, soluble CR1 constructs can be obtained.

Expression vectors containing CR1 gene inserts can be identified bythree general approaches: (a) DNA-DNA hybridization, (b) presence orabsence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous tothe inserted CR1 gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain “marker” gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes into the vector. For example, if the CR1 gene isinserted within the marker, gene sequence of the vector, recombinantscontaining the CR1 insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the foreign gene product expressed by therecombinant. Such assays can be based on the physical, immunological, orfunctional properties of the gene product.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity.

In a particular embodiment detailed in the examples of the presentinvention, CDM8 vectors with an CR1 cDNA insert can be transfected intoCOS cells, in which the CR1 cDNA insert is expressed to produce the CR1protein. In other particular embodiments detailed in the examplessections infra, CDM8 vectors with a CR1 cDNA insert corresponding to aportion of the CR1 coding region can be transfected into COS cells,where the CR1 or fragment is expressed. Per yet another example, infra,truncated, soluble CR1 molecules can be expressed in mammalian cells byuse of expression vectors such as the PTCS vectors described in Section11.3.1. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes thechimeric gene product in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered CR1 protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the desired modification and processingof the expressed heterologous protein. For example, in one embodiment,expression in a bacterial system can be used to produce anunglycosylated CR1 protein with the deduced amino acid sequence of FIG.1. Expression in yeast will produce a glycosylated product. In anotherembodiment, mammalian COS cells can be used to ensure “native”glycosylation of the heterologous CR1 protein. Furthermore, differentvector/host expression systems may effect processing reactions such asproteolytic cleavages to different extents. Many such variouslyprocessed CR1 proteins can be produced and are within the scope of thepresent invention.

In a preferred embodiment of the invention, large scale production ofsoluble CR1 molecules may be carried out as described infra in Section12.1 et seq.

5.3. Identification and Purification of the Expressed Gene Product

Once a recombinant which expresses the CR1 gene is identified, the geneproduct should be analyzed. This can be achieved by assays based on thephysical, immunological, or functional properties of the product.

The CR1 proteins may be isolated and purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, and sizingcolumn chromatography, high pressure liquid chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

In a preferred aspect of the invention detailed in the examples infra,large quantities of soluble CR1 can be purified by procedures involvingHPLC (see Section 12.2 et seq.). As described infra, large-scaleproduction of purified CR1 can be achieved by using an expression systemwhich produces soluble CR1 as starting material, thus eliminating therequirement of solubilizing membrane-bound CR1 with detergents. Thereduction of fetal calf serum concentrations in the bioreactor culturesand/or the use of alternative culture medias in these cultureseliminates the need to remove high concentrations of extraneous proteinsfrom the soluble CR1-containing starting material during subsequentpurification. Either cation HPLC or a combination of cation HPLCfollowed by anion exchange HPLC can be used for purification in thispreferred aspect. Substantially pure soluble CR1 in high yield can thusbe achieved in only one or two steps.

Alternatively, once a CR1 protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller, M., et al.,1984, Nature 310:105-111).

In particular embodiments of the present invention, such CR1 proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods, include but are not limited to those containing, as a primaryamino acid sequence, all or part of the amino acid sequencesubstantially as depicted in FIG. 1, including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Nonconservativesubstitutions can also result in functionally equivalent proteins.

In one embodiment, substitutes for an amino acid within the CR1 sequencemay be selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand nethionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention are CR1proteins which are differentially modified-during or after translation,e.g., by glycosylation, proteolytic cleavage, etc.

In an example of the invention detailed infra, cloned recombinant CR1expressed by transfected cells was shown to be indistinguishable fromthe F allotype of erythrocytes by SDS-PAGE (FIG. 14), capable ofmediating the binding of sheep erythrocytes bearing either C4b or C3b,and able to reproduce the ligand specificity of CR1 (FIG. 13), andexhibit factor I co-factor activity for cleavage of the alphapolypeptide of C3(ma) (FIG. 15).

5.4. Structure of the CR1 Gene and Protein

The structure of the CR1 gene and protein can be analyzed by variousmethods known in the art, including but not limited to those describedinfra.

5.4.1. Genetic Analysis

The cloned DNA or cDNA corresponding to the CR1 gene can be analyzed bymethods including but not limited to Southern hybridization (Southern,E. M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (seee.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098),restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.), and DNA sequence analysis. The stringency of the hybridizationconditions for both Southern and Northern hybridization can bemanipulated to ensure detection of nucleic acids with the desired degreeof relatedness to the specific CR1 probe used. For example,hybridization under low stringency conditions with a probe containingCR1 gene sequences encoding LHR-B and LHR-C, can be used to detect CR2nucleic acid sequences.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure or the CR1 gene. In a particular embodiment, cleavagewith restriction enzymes can be used to derive the restriction map shownin FIG. 2, infra. Restriction maps derived by restriction endonucleasecleavage can be confirmed by DNA sequence analysis.

DNA sequence analysis can be performed by any techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Math. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463),a or use of aautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.). The cDNA sequence of the CR1 gene comprises the a sequencesubstantially as depicted in FIG. 1, and, described in Sections 6 and 7,infra.

5.4.2. Protein Analysis

The amino acid sequence of the CR1 protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer. The amino acidsequence of a representative CR1 protein comprises the sequencesubstantially as depicted in FIG. 1, and detailed in Section 6, infra.As described infra, all of the coding sequence of the F allotype CR1 hasbeen cloned and, after cleavage of the signal peptide of 41 amino acids,the mature receptor contained 1998 amino acids including anextracellular domain of 1930 residues that forms 30 SCRs,, 28 10), ofwhich are organized into LHRs-A, -B,, -C and -D, (FIG. 10) a singlemembrane spanning domain of 25 amino acids and a relatively shortcytoplasmic domain of 43 amino acids.

Among the C3/C4 binding proteins that contain multiple SCRs, CR1 isunique in having groups of SCRs organized into LHRs. Comparison of thefour LHRs of CR1 reveals that each is a composite of four types of SCRs:types a, b, c and d (FIG. 19). For example, the sequences; of SCR-1 and-2 of LHR-A are only 62%, 62% and 57% identical to the first two SCRs ofLHR-B, -C and -D, respectively. However, SCR-3 through SCR-7 differ fromthe corresponding SCRs of LHR-B at only a single position, and SCR-3 and-4 differ from those of LHR-C at only three positions (FIG. 10). Thus,some of the type “a” SCRs of LHR-A are also present in LHR-B and -C. Thefirst two SCRs of LHR-B, which differ from those of LHR-A, are 99%identical with the corresponding SCRs of LHR-C, so that LHR-B and -Cshare the type “b” SCR at these positions. The fifth, sixth and seventhSCR of LHR-C are only 77% identical to the type “a” SCRs in LHR-A and -Bat these positions, and are considered as type “c” SCRs., The firstthrough fourth SCRs of LHR-D are relatively unique and are type “d”,while the fifth through seventh SCRs are approximately 93% identical tothe “c” type found in LHR-C.

The CR1 protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identifythe hydrophobic and hydrophilic regions of the CR1 protein and thecorresponding regions of the gene sequence which encode such regions. Ahydrophilicity profile of the COOH-terminus of the CR1 protein isdepicted in FIG. 5.

Secondary structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to predict regions of CR1 thatassume specific secondary structures.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.5. CR1-Related Derivatives, Analogues, and Peptides

The production and use of derivatives, analogues, and peptides relatedto CR1 are also envisioned, and within the scope of the presentinvention. Such derivatives, analogues, or peptides which have thedesired immunogenicity or antigenicity can be used, for example, inimmunoassays, for immunization, therapeutically, etc. Such moleculeswhich retain, or alternatively inhibit, a desired CR1 property, e.g.,binding of C3b or C4b, regulation of complement activity, or promotionof immune stimulation or phagocytosis, etc., can be used as inducers, orinhibitors, respectively, of such property.

The CR1-related derivatives, analogues, and peptides of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, the cloned CR1 gene can be modified by any of numerousstrategies known in the art (Maniatis, T., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). The CR1 sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro (see Section 8, infra). Inthe production of the gene encoding a derivative, analogue, or peptiderelated to CR1, care should be taken to ensure that the modified generemains within the same translational reading frame as CR1,uninterrupted by translational stop signals, in the gene region wherethe desired CR1-specific activity is encoded.

Additionally, the CR1 gene can be mutated in vitro or in vivo, to createand/or destroy translation, initiation, and/or termination sequences, orto create variations in coding regions and/or form new restrictionendonuclease sites or destroy preexisting ones, to facilitate further invitro modification. Any technique for mutagenesis known in the art canbe used, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C. , et al. , 1978, J. Biol. Chem. 253:6551),use of TAB® linkers (Pharmacia), etc.

Manipulations of the CR1 sequence may also be made at the protein level.Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited to specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄;acetylation, formulation, oxidation, reduction; metabolic synthesis inthe presence of tunicamycin; etc.

In addition, analogues and peptides related to CR1 can be chemicallysynthesized. For example, a peptide corresponding to a portion of CR1which mediates the desired activity (e.g., C3b and/or C4b binding,immune stimulation, complement regulation, etc.) can be synthesized byuse of a peptide synthesizer.

5.6. Uses of CR1

5.6.1. Assays and Diagnosis

CR1 proteins, analogues, derivatives, and subsequences thereof, andanti-CR1 antibodies, have uses in assays and in diagnostics. Themolecules of the invention which demonstrate the desired CR1 property orfunction can be used to assay such property or function. For example,CR1 proteins or fragments thereof, which exhibit binding of C3b and/orC4b, in free and/or in complex forms, can be used in assays to measurethe amount of such substances in a sample, e.g., a body fluid of apatient.

In a specific embodiment, full-length CR1 or a CR1 deletion mutantexpressed on the cell surface (e.g., those described in Section 8,infra) having the ability to bind C3b (e.g., see Table II, Section 9,infra), iC3b or C4b (e.g., see Table II) can be used in assays tomeasure the levels of C3b, iC3b, or C4b, respectively, in a sample. Inanother embodiment, a CR1 protein or fragment thereof which isconstructed by recombinant DNA technology to lack a transmembranesequence, and is thus secreted, can be used.

In a particular embodiment, such a measurement of C3b and/or C4b can berelied on as an indication of complement activity, and can be useful inthe diagnosis of inflammatory and immune system disorders. Suchdisorders include but are not limited to tissue damage due to burn- ormyocardial infarct-induced trauma, adult respiratory distress syndrome(shock lung), autoimmune disorders such as rheumatoid arthritis,systemic lupus erythematosus, and other diseases or disorders involvingundesirable or inappropriate complement activity (see, e.g., Miescher,P. A. and Muller-Eberhard, H. J., ads., 1976, Text Book ofImmunopathology, 2d Ed., Vols. I and II, Grune and Stratton, New York;Sandberg, A. L., 1981, in,Cellular Functions in Immunity andInflammation, Oppenheim, J. J. et al., eds., Elsevier/North Holland, NewYork, p. 373; Conrow, R.B. et al., 1980, J. Med. Chem. 23:242; Regal, J.F. and Pickering, R. H., 1983, Int. J. Immunopharmacol. 5:71; Jacobs, H.S., 1980, Arch. Pathol. Lab. Ned. 104:617).

The CR1 protein and fragments thereof containing an epitope have uses inassays including but not limited to immunoassays. The imunoassays whichcan be used include but are not limited to competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and immunoelectrophoresis assays, to name but a few.

CR1 genes and related nucleic acid sequences and subsequences, can beused in hybridization assays. Such hybridization assays can be used tomonitor inflammatory or immune responses associated with CR1 expression,to diagnose certain disease states associated with changes in CR1expression, to determine the CR1 allotype of a patient, and to detectthe presence and/or expression of the CR1 gene and related genes (e.g.,CR2).

5.6.2. Therapy

The CR1 protein and fragments, derivatives, and analogues thereof can betherapeutically useful in the modulation of functions mediated by CR1.Such functions include but are not limited to binding of C3b and/or C4b,in free or in complex forms, promotion of phagocytosis, complementregulation, immune stimulation, etc. Effective doses of the CR1 proteinsand related molecules of the invention have therapeutic value for manyof the diseases or disorders associated with such functions, such asimmune or inflammatory disorders (e.g., those described supra in Section5.6.1). For example, full-length CR1 or fragments thereof and relatedmolecules which exhibit the desired activity can have therapeutic usesin the inhibition of complement by their ability to act as a factor Icofactor, promoting the irreversible inactivation of complementcomponents C3b or C4b (Fearon, D. T., 1979, Proc. Natl. Acad. Sci.U.S.A. 76:5867; Iida, K. and Nussenzweig, V., 1981, J. Exp. Med. 153:1138), and/or by the ability to inhibit the alternative or classical C3or C5 convertases.

In a specific embodiment of the invention, an expression vector can beconstructed to encode a CR1 molecule which lacks the transmembraneregion (e.g., by deletion carboxy-terminal to the arginine encoded bythe most C-terminal SCR), resulting in the production of a soluble CR1fragment. In one embodiment, such a fragment can retain the ability tobind C3b and/or C4b, in free or in complex forms. In a particularembodiment, such a soluble CR1 protein may no longer exhibit factor Icofactor activity. The soluble CR1 product can be administered in vivoto a patient, so that the soluble CR1 can effectively compete outbinding of the C3b and/or C4b to the.-native cell-surface CR1, thusblocking cell-surface CR1 factor I cofactor activity, and increasingcomplement activity.

After C3b has covalently attached to particles and soluble immunecomplexes, the inactivation of C3b by protoolytic processing into iC3band C3dg has two biologic consequences: preventing excessive activationof the complement system via the amplification pathway, and formation ofligands that can engage receptors other than CR1. The iC3b fragmentcannot bind factor B so that conversion to this state blocks additionalcomplement activation via the alternative pathway amplification loop.However, iC3b can be bound by CR1 and CR3, the two complement receptorsthat mediate phagocytosis by myelomonocytic cells. Therefore, theprimary biologic consequence of C3b to iC3b conversion is cessation ofcomplement activation without interference with CR1- and CR3-mediatedclearance of the C3-coated complex. In contrast, the additionalconversion of iC3b to C3dg creates a fragment that interacts only withCR2 and not with CR1 and CR3. This circumstance limitscomplement-dependent binding of the C3dg-bearing complex to call typesexpressing CR2, which include B lymphocytes, follicular dendritic cellsand perhaps epitholial cells of the dermis, and diminishes or excludesinteraction with phagocytic cell types. The biologic consequence of thisaltered pattern of cellular association would be targeting of theC3dg-bearing complexes to cells involved in the afferent phase of theimmune response rather than to calls involved in clearance anddegradation of particles and complexes. Therefore, CR1 molecules may beused therapeutically not only to affect the clearance process, but alsoin the targeting of complexes to the CR2-bearing cell types thatparticipate in antigen presentation and antibody production.

In an alternative embodiment, a CR1 protein or fragment thereof whichcan bind C3b or C4b, and/or retains; the ability to inhibit thealternative or classical C3 or C5 convertases, or retains factor Icofactor activity, can be used to promote complement inactivation. Insuch an embodiment, the CR1 protein or fragment can be valuable in thetreatment of disorders which involve undesirable or inappropriatecomplement activity (e.g., shock lung, tissue damage due to burn orischemic heart conditions, autoimmune disorders, inflammatoryconditions, etc.).

In a specific embodiment detailed in the examples Sections 11-14 infra,a soluble CR1 molecule can be expressed which retains a desiredfunctional activity, as demonstrated, e.g., by the ability to inhibitclassical complement-mediated hemolysis, classical C5a production,classical C3a production, or neutrophil oxidative burst in vitro. In aparticular embodiment, such a soluble CR1 molecule can be used to reduceinflammation and its detrimental effects, or to reduce myocardialinfarct size or prevent reperfusion injury, etc. Such CR1 moleculesuseful for in vivo therapy may be tested in various model systems knownin the art, including but not limited to the reversed passive Arthrusreaction. (see Section 14.1) and a rat myocardial infarct model (seeSection 14.3).

In another-embodiment of the invention, a fragment of CR1, or ananalogue or derivative thereof, which is shown to inhibit a desired CR1property or function, can be used to prevent or treat diseases ordisorders associated with that function.

Various delivery systems are known and can be used for delivery of CR1and related molecules, e.g., encapsulation in liposomes, expression byhematopoietic stem cell progeny in gene therapy, etc. Other methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.

6. EXAMPLE The Cloning and Sequencing of The Human C3b/C4b Receptor(CR1)

In the examples detailed herein, we describe the cloning and nucleotidesequence of 5.5 kilobase pairs (kb) of the CR1 coding region(Klickstein, L. B., et al., 1987, J. Exp. Med. 165:1095-1112).

Ten overlapping CR1 cDNA clones that span 5.5 kb were isolated from atonsillar library and sequenced in whole or in part. A single long openreading frame beginning at the 5′ end of the clones and extending 4.7 kbdownstream to a stop codon was identified. This sequence represents ˜80%of the estimated 6 kb of coding sequence for the F allotype of CR1.Three tandem, direct, long homologous repeats (LHRs) of 450 amino acidswere identified. Analysis of the sequences of tryptic peptides providedevidence for a fourth LHR in the F allotype of CR1. Amino acid identitybetween the LHRs ranged from 70% between the first and third repeats to99% between the NH₂-terminal 250 amino acids of the first and secondrepeats. Each LHR comprises seven short consensus repeats (SCRs) of60-70 amino acids that resemble the SCRs of other C3/C4 bindingproteins, such as complement receptor type 2, factors B and H, C4binding protein, and C2. Two additional SCRs join the LHRs to a singlemembrane-spanning domain of 25 amino acids: thus, the F allotype of CR1probably contains at least 30 SCRs, 23 of which have been sequenced.Each SCR is predicted to form a triple loop structure in which the fourconserved half-cystines form disulfide linkages. The linear alignment of30 SCRs as a semi-rigid structure would extend 1,140 Angstroms from theplasma membrane and might facilitate the interaction of CR1 with C3b andC4b located within the interstices of immune complexes and microbialcell walls. The COOH-terminal-cytoplasmic domain of 43 residues containsa six amino acid sequence that is homologous to the sequence in theepidermal growth factor receptor that is phosphorylated by proteinkinase C.

6.1. Materials and Methods

6.1.1. Isolation and Sequence of CR1 Tryptic Peptides

CR1 was purified from washed human erythrocyte membranes by sequentialMatrex Rid A and YZ-1 monoclonal antibody affinity chromatography (Wong,W. W., et al., 1985, J. Immunol. Methods 82:303). Tryptic peptides wereprepared and isolated by sequential gradient and isocratic reverse-phaseHPLC (high performance liquid chromatography) as described (Wong, W. W.,et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7711). Tryptic peptideanalysis was performed with a 470A Protein Sequencer (AppliedBiosystems, Inc., Foster City,, Calif.), and analysis of eachdegradative cycle was achieved using a 120 PTH-amino acid analyzer(Applied Biosystems, Inc.).

6.1.2. Isolation of cDNA Clones and Genomic Clones

A cDNA library was constructed in λgt11 from human tonsilar poly(A)⁺ RNAas described (Wong, W. W., et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:7711). By RNA blot hybridization, the tonsil donor was homozygous forthe F allele of CR1 (id.). The cDNA was selected on an agarose gel toinclude fractions between 2 and 7 kb before the cloning steps. Theinitial complexity of the library was 4.5×10⁶ recombinants per 100 ngcDNA and the library was amplified in Escherichia coli strain Y1088. Thelibrary was screened (Maniatis, T., et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) with CR1 probes, CR1-1 (ATCC accession nos. 57330 (E. colicontaining CR1-1 plasmid), 57331 (purified CR1-1 DNA)) and CR1-2 (Wong,W. W., et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7711), that hadbeen radiolabeled to a specific activity of 2-8×10⁸ cpm/μg by nicktranslation. Hybridization was performed in 50% formamide, 5x SSC (1xSSC: 15 mM sodium citrate, 150 mM sodium chloride) at 43° C. and filterswere washed at 60° C. in 0.2x SSC, conditions that do not allow thedetection of CR2 cDNA clones (Weis, J. J., et al., 1986, Proc. Natl.Acad. Sci. U.S.A. 83:5639). Positive clones were plaque-purified twicebefore restriction mapping and DNA sequence analysis.

A genomic library was constructed in EMBL-3 with 15-20 kb fragmentsproduced by partial digestion of human leukocyte DNA with Sau3AI. Theinitial complexity was 1.2×10⁶, and the library was amplified in E. colistrain P2392. The library was also screened with the cDNA probes CR1-1and CR1-2 (Wong, W. W., et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:7711).

6.1.3. DNA Sequence Analysis

Restriction fragments of the cDNA clones were subcloned in M13mp18 orM13mp19 and sequenced by the dideoxynucleotide chain termination method(Sanger, F., et al., 1977, Proc. Natl. Acad. Sci. U.S,A. 74:5463). Someclones were sequenced in whole or in part by first creating ordereddeletion mutants using exonuclease III (Henikoff, S., 1984, Gene28:351). Each region was sequenced on both strands and in most castseach region was sequenced on M13 subcones constructed from twoindependently isolated cDNA clones (FIG. 2). Sequence data were analyzedwith the University of Wisconsin Genetics Computer Group package(Madison, Wis.).

6.2. Results

6.2.1. Nucleotide Sequence of the CR1 Gene

A size-selected tonsillar cDNA library was screened with the CR1-1 andCR1-2 probes obtained from the CR1 cDNA clone, λT8.3 (Wong, W. W., etal., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7711). Fifteen positivephage were identified out of 1.5×10⁶ recombinants and 13 of theserepresented distinct clones. Ten were restriction mapped and sequencedin whole or in part by the dideoxynucleotide chain termination method.The cDNA clones were aligned on the basis of overlapping sequenceidentity (FIG. 2) and were found to span 5.5 kb (FIGS. 3A-3D). A singlelong open reading frame was identified beginning at the 5′ and of thecDNA clones and extending 4.7 kb downstream to a stop codon. The codingsequence for CR1 in this library is expected to be 6 kb, based on anestimated 220,000 dalton molecular weight for the nonglycosylatedreceptor (Wang, W. W., et al., 1983, J. Clin. Invest. 72:685). Thus,these clones span ˜80% of the estimated coding sequence.

Clones T49.1 and T55.1 contain coding sequence at their 5a ends,indicating that additional 5′ coding and noncoding sequences remain tobe identified. In the 3′ region, the overlapping clones, T8.2, T43.1 andT87.1, contain the transmembrane and cytoplasmic regions encoded by anidentical sequence in each clone. The clone extending most 3′, T8.2,contains 807 bp of untranslated sequence without a poly(A) sequence.Clone T8.3 contains a 91-bp deletion of nucleotides 1,406-1,497 andclone T40.1 contains a 9-bp deletion of nucleotides 1,498-1,507 relativeto the sequences found in clones T6.1 and T55.1. These deletionsoccurred in regions having sequences homologous to 5′ splice sites andmay represent splicing errors in the mRNA. Clones T49.1 and T55.1.contain a 110 bp insertion between nucleotides 147 and 148 of the openreading frame (FIGS. 3A-3D). This sequence is judged to be a portion ofan intron because it did not hybridize to blots of tonsillar poly(A)⁺RNA, it contains a 5′ splice site (Breathnach, R., et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:4853) (FIGS. 3A-3D), it is flanked by cDNAsequences in CR1 genomic clones, and it shifts the reading frame. CloneT9.4 contains 0.88 kb of intervening sequence at the 3′ end that doesnot hybridize to blots of tonsillar poly(A)⁺ RNA.

6.2.2. Analysis of the Nucleotide and Amino Acid Sequence of CR1

Dot matrix analysis of the nucleotide sequence of CR1 (FIGS. 3A-3D)revealed two types of internal homologies (FIG. 4). The first type ofinternal homology is represented by the bold, uninterrupted lines thatindicate the presence of three tandem, direct, highly homologous repeatsof 1.35 kb. These nucleotide sequences encode the long homologousrepeats (LHRs) of CR1. The second type of repeat is represented by thedashed parallel lines that indicate regions of lesser homology. Thesesequences occur every 190-210 nucleotides and encode the short consensusrepeats (SCRs) of CR1.

The amino acid sequence deduced from the cDNA sequence is presented inFIG. 5B and the three LHRs, designated LHR-B, LHR-C and LHR-D, arealigned to demonstrate their homology. LHR-B extends from residue 1through residue 438, LHR-C corresponds to residues 439-891, and LHR-Dextends from residue 892 through 1,341. Residues 451-694 of LHR-C are99% identical to residues 1-244 of LHR-B, but only 61% identical to thecorresponding residues of LHR-D. In contrast, residues 695-891 of LHR-Care 91% identical to residues 1,148-1,341 of LHR-D but only 76%identical to the corresponding region of LHR-B. Thus, LHR-C appears tobe a hybrid that comprises sequences most homologous to the first halfof LHR-B and the second half of LHR-D. The LHRs are followed by two SCRsthat are not repeated, a 25 residue hydrophobic segment and a 43 aminoacid COOH-terminal region with no sequence homology to the SCRs (FIG.5B).

The 5′ 1.3 kb of the CR1 coding sequence represents a fourth LHR, LHR-A(see FIGS. 1A-H, supra, and Section 7, infra). This conclusion wassupported by analysis of tryptic peptides of erythrocyte CR1. Tentryptic peptides have sequences identical to the amino acid sequencesderived from the cDNA clones (Table I).

TABLE I CR1 TRYPTIC PEPTIDES FOUND IN THE DERIVED AMINO ACID SEQUENCE*Pep- tide Residue Numbers Num- in the Derived ber Amino acid sequenceSequence 66 VDFVCDEGFQLKGS-A 330-345 28 GAASL----QG-WSPEAP  732-749,1,185-1,202 49 ------------IFC-NP-AIL  805-826, 1,258-1,279 35CQALNKWEPELPSCSR  228-243, 678-693 41c DKDNFSPGQEVFYSCEPGYDLR 260-28134b AV-YTCDPHPDRGTSFDLIGESTIR 393-417 44d VCQPPPEILHG  694-704,1,147-1,157 54d VFELVGEPSIYCTSNDDQVGIWSGPAPQ  152-179, 602-629 57bYECRPEYYGRPFS  19-31, 469-481 39b LIGHSSAECILSGNAA  85-100 *Trypticpeptides from human erythrocyte CR1 found in the derived amino acidsequence. The number ranges in the right-hand column indicate thelocation of the peptide in the derived amino acid sequence. Each dash inpeptides 66, 28 and 49 indicates multiple residues were identified atthat cycle. The dash in peptide 34b indicates no residue was identifiedat that cycle.

Each LHR comprises seven 60-70 amino acid SCRs that characterize thefamily of C3 and C4 binding proteins (C4bp) (FIG. 6A). Maximal homologybetween the 23 SCRs of CR1 was observed by introducing spaces in thealignment of the sequences (FIG. 6A). Altogether, 29 of the average 65residues in each repeat are conserved. There are six residues that arepresent in all SCRs: the four half-cystines that are in similar relativepositions suggesting that each may be involved in a critical disulfidelinkage, and the tryptophan and the second glycine after the secondhalf-cystine (FIG. 6A). Secondary structure analysis of the sequencesbetween the invariant half-cystines using the algorithm of Chou andFasman (Chou, P. Y. and Fasman, G. D., 1974, Biochemistry 13:222)predicted high probability β-turn formation and low probability α-helixformation. Sequence analysis of two CR1 genomic clones, 2.38 (FIG. 6B).and 2.46, indicates that SCR-14 (FIG. 6A) is encoded by a single exonand that the COOH-terminus of SCR-23 corresponds to the end of an exon.Thus, the SCRs of CR1 may be encoded by separate exons as has been shownfor the SCRs of factor B (Campbell, R. D. and Bentley, D. R., 1985,Imunol. Rev. 87:19) and of the IL-2-R (Leonard, W. J., et al., 1985,Science 230:633).

The consensus sequence of the CR1 SCRs is compared with the SCRs of theother members of the superfamily having this characteristic structure(FIG. 7). These members include not only proteins having C3/C4 bindingfunction, CR2 (Weis, J. J., et al., 1986, Proc. Natl. Acad. Sci. U.S.A.83:5639), C4bp (Chung, L. P., at al., 1985, Biochea. J. 230:133), factorH (Kristensen, T., et al., 1986, J. Immunol. 136:3407), factor B(Morley, B. J. and Campbell, R. D., 1984, EMBO J. 3:153; Mole, J. E., etal., 1984, J. Biol. Chem. 259:3407), and C2 (Bentley, D. R. and Porter,R. R., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:1212;

Gagnon, J., 1984, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 306:301),but also the proteins not known to have this function, such as theinterleukin-2 receptor (Leonard, W. J., et al., 1985, Science 230:633),β₂-glycoprotein I (Lozier, J., et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:3640), Clr (Leytus, S. P., et al., 1986, Biochemistry25:4855), haptoglobin a chain (Kurosky, A., et al., 1980, Proc. Natl.Acad. Sci. U.S.A. 77:3388), and factor XIItb (Ichinose, A., et al.,1986, Biochemistry 25:4633). The half-cystine residues are invariant inthe SCRs of all proteins, except haptoglobin which lacks the thirdhalf-cystine. The tryptophan is also invariant with the exception of thefifth SCR in β₂-glycoprotein I and two of the repeats in factor XIIIb.Other residues that are conserved but not present in 15 each SCR tend tocluster about the half-cystines. There is only one free thiol group infactor B and C2 (Christie, D. L. and Gagnon, J., 1982, Biochem. J.201:555; Parkes, C., et al., 1983, Biochen. J. 213:201), and in the SCRsof glycoprotein I, the first half-cystine is disulfide-linked to thethird and the second to the fourth (Lozier, J., et al., 1984, Proc.Natl. Acad. Sci. U.S.A. 81:3640).

In the derived amino acid sequence of CR1, there are 17 potential sitesfor N-linked oligosaccharides and all of them are in the extracellularregion (FIG. 6A). Molecular weight differences between CR1 synthesizedin the presence and absence of tunicamycin (Lublin, D. M., et al., 1986,J. Biol. Chem. 261:5736) and analysis of glucosamine content (Sim, R.B., 1985, Biochem. J. 232:883) suggest the presence of only 6-8 N-linkedcomplex oligosaccharides, indicating that all potential sites are notused. For example, the asparagine at residue 263 of the derived aminoacid sequence (FIG. 5B) was identified in peptide 41c (Table I),indicating absence of glycosylation at this site. In contrast, theunidentified amino acid in peptide 34b probably corresponds to aglycosylated asparagine at residue 395.

The only nonrepetitive CR1 sequences identified in the 5.5 kb of cDNAare located in the COOH-terminal region. A secondary structure analysisof this region identifies a single 25-residue putative membrane-spanningsegment having strong hydrophobic character and high potential fora-helix formation (FIG. 5C). This sequence is immediately followed byfour positively charged residues, a characteristic of many membraneproteins. The presumed cytoplasmic region of CR1 comprises 43 residuesand contains a six amino acid sequence, VHPRTL, which is homologous tothe sequence VRKRTL, a site of protein kinase C phosphorylation in theepidermal growth factor (EGF) receptor and the erbB oncogene product(Hunter, T., et al., 1984, Nature 311:480; Davis, R. J. and Czech, M.P., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:1974). There are no tyrosineresidues in the cytoplasmic region of tonsillar CR1.

6.3. Discussion

Approximately 80% of the primary structure of the F allotype of CR1 hasbeen obtained by sequencing overlapping cDNA clones. The most unusualstructural feature of CR1 observed in this analysis is the presence oftandem, direct LHRs of 450 amino acids, four of which are predicted tooccur in the F allotype of CR1 that has an estimated polypeptide chainlength of 2,000 residues (Wong, W. W., et al., 1983, J. Clin. Invest.72:685; Sim, R. B., 1985, Biochem. J. 232:883). Three of the LHRs havebeen cloned and sequenced while evidence for the existence of the fourthwas provided by the analysis of tryptic peptides. Each LHR is comprisedof seven SCRs which are the basic structural elements of other C3/C4binding proteins. The conservation of the four half-cystines per SCR,the probable involvement of the first and third and the second andfourth half-cystines in disulfide linkages (Lozier, J., et al., 1984,Proc. Natl. Acad. Sci. U.S.A. 81:3640) and the presence of conservedamino acids such as proline, glycine and asparagine which are frequentlyfound in β-turns (Rose, G. D., et al., 1985, Adv. Protein Chem. 37:1)lead to the proposal that an SCR forms a triple loop structuremaintained by disulfide linkages (FIG. 8B). This role for thehalf-cystine residues is supported by the finding that mildlytrypsin-treated CR1 (Sim, R.B., 1985, Biochem. J. 232:883) and factor H(Sim, R. B. and DiScipio, R. G., 1982, Biochem. J. 205:285) migrate asintact molecules when analyzed by SDS-polyacrylamide gel electrophoresis(PAGE) under non-reducing conditions and as multiple tryptic fragmentsafter reduction.

This series of tandemly repeated SCRs in predicted to form an elongatedstructure (FIG. 8A) as has been proposed for factor H and for eachsubunit of human C4bp (Sim, R. B. and DiScipio, R. G., 1982, Biochem. J.205:285; Whaley, K. and Ruddy, S., 1976, J. Exp. Med. 144:1147;Dahlback, B., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3461).Electron microscopic studies of the subunits of C4bp have indicateddimensions of 300×30 Angstroms (Dahlback, B., et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:3461). As each subunit is composed of eight SCRs(Chung, L. P., et al., 1985, Biochem. J. 230:133), an individual SCR iscalculated to be 38×30 Angstroms. Assuming that the SCRs of CR1 havesimilar dimensions and that the F allotype has 30 SCRs, the receptorcould extend as much as 1,140 Angstroms from the cell membrane.Consistent with this prediction of CR1 structure in the earlier findingthat ferritin-labeled antibody bound to CR1 on neutrophils wasfrequently 500 Angstroms from the outer leaflet of the plasma membrane(Abrahamson, D. R. and Fearon, D. T., 1983, Lab. Invest. 48:162). Suchan elongated structure of CR1 would facilitate the interaction ofreceptor-bearing cells with C3b that has covalently bound to relativelyinaccessible sites within immune complexes and microbial cell surfaces.

The-finding that the SCR is the major, and perhaps only,extracytoplasmic element of CR1 provides structural evidence for a closerelationship between the receptor and factor H and C4bp, two plasmaproteins that are exclusively or predominantly composed of SCRs (Chung,L. P., et al., 1985, Biochem. J. 230:133; Kristensen, T., et al., 1986,J. Immunol. 136:3407). CR1 was initially isolated as an erythrocytemembrane protein having factor H-like activity after detergentsolubilization (Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A.76:5867), and it was found subsequently to have the regulatory functionsof factor H and C4bp when residing on the plasma membrane (Iida, K. andNussenzweig, V., 1981, J. Exp. Med. 153:1138). By analysis of theinheritance of structural polymorphisms of CR1, factor H, and C4bp,, thegenes encoding these three proteins were shown to be linked (de Cordoba,R., et al., 1985, J. Exp. Med. 161:1189), and the locus for this linkagegroup and for the structurally related receptor, CR2, have been shownrecently by in situ hybridization and by the analysis of somatic cellhybrids to be on the long arm of chromosome 1, band q32 (Weis, J. H., etal., 1987, J. Immunol. 138:312). Before the present study, the onlyevidence for a structural relationship between these proteins was asignificant similarity in their amino acid compositions (Wong, W. W., etal., 1985, J. Immunol. Methods 82:303). Therefore, the present findingof at least 23 SCRs in CR1 constitutes the direct and formaldemonstration of a structural relationship of the receptor with factor Hand C4bp (Chung, L. P., et al., 1985, Biochem. J. 230:133; Kristensen,T., et al., 1986, J. Immunol. 136:3407), proteins with similarfunctions, and with the Ba and C2b fragments of factor B and C2 (Morley,B. J. and Campbell, R. D., 1984, EMBO J. 3:153; Hole, J. E., et al.,1984, J. Biol. Chem. 259:3407; Bentley, D. R. and Porter, R. R., 1984,Proc. Natl. Acad. Sci. U.S.A. 81:1212; Gagnon, J., 1984, Philos. Trans.R. Soc. Lond. B Biol. Sci. 306:301), components that form enzymaticcomplexes with C3b and C4b, respectively. However, the SCR is also foundin several noncomplement proteins (Campbell, R. D., and Bentley, D. R.,1985, Immunol. Rev. 87:19; Lozier, J., et al., 1984, Proc. Natl. Acad.Sci. U.S.A. 81:3640; Leytus, S. P., et al., 1986, Biochemistry 25:4855;Kurosky, A., et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:3388;Ichinose, A., et al., 1986, Biochemistry 25:4633) (FIG. 7), indicatingthat it does not necessarily represent a C3/C4 binding structure.

Among the proteins having SCRs, CR1 is unique in having organized thisbasic structure and genetic unit into the higher order structural unitof the LHR. Analysis of a 14.5 kb BamHI fragment of genomic DNA that isassociated with expression of the S allotype has suggested that at leastone repeating genomic unit in CR1 is an extended segment of DNAcontaining the exons encoding at least five SCRs and their flankingintrons (Wong, W. W., et al., 1986, J. Exp. Med. 164:1531). Thesestudies have also suggested that the S allele contains an additionalcopy of this genomic unit compared with the number present in the Fallele. This observation, combined with a tryptic peptide mapping study(Nickells, M. W., at al., 1986, Mol. Immunol. 23:661) and the presentfinding that an LHR represents a peptide of ˜40-50 kD allows us topredict the presence in the S allotype (290 kD) of an additional LHRrelative to the, estimate of four LHRs in the F allotype (250,000daltons molecular weight).

In addition to providing evidence for duplication events, the sequencesof the LHRs also suggest that conversion events have occurred within theCR1 gene. LHR-B and -D are 67% identical to each other throughout theirlength, whereas LHR-C is 99% identical to LHR-B in the NH₂-terminal fourSCRs and 91% identical to LHR-D in the COOH-terminal three SCRs. Thisorganization could not have occurred by a single recombinational eventbetween identical parental alleles in the origin of this hybrid LHR.Rather, the hybrid LHR may have arisen by gene conversion (Atchison, M.and Adesnik, M., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:2300) in whichsequences in an LHR-C precursor were replaced by sequences present inLUR-B or LUR-D. The near complete identity and precise alignment ofhomologous sequences in these LHRs (FIG. 5B) also may have beenmaintained by a mechanism involving gene conversion. Analysis of theextent of homology between intervening sequences of those segments ofthe CR1 gene encoding the LHRs should determine whether gene conversionor selection based on functional constraints have strictly limitedsequence divergence.

Although a previous study suggested that CR1 is monovalent (Wilson, J.G., et al., 1982, N. Engl. J. Med. 307:981), each LHR might represent asingle C3b/C4b binding domain, which would make the receptor multivalentand adapted for the binding of complexes bearing multiple molecules ofC3b and C4b. Alternatively, distinct LHRs might be responsible forbinding C3b and C4b, respectively (see Section 9, infra), providing astructural basis for the combination of factor H and C4bp activities inCR1. Finally the LHRs of CR1 may represent structural domains that serveto extend CR1 from the plasma membrane, as suggested by the proposedstructural model (FIG. 8A), and SCRs at the NH₂-terminal region bind C3band C4b, as has been found for factor H (Sim, R. B. and DiScipio, R. G.,1982, Biochem. J. 205:285; Alsenz, J., et al., 1984, Biochem. J.224:389).

Activation of protein kinase C by phorbol esters induces phosphorylationof CR1 in neutrophils, monocytes, and eosinophils (Changelian, P. S. andFearon, D. T., 1986, J. Exp. Med. 163:101) and the CR1 cytoplasmicdomain of 43 amino acids has a sequence that is homologous to a sitethat is phosphorylated by protein kinase C in the epidermal growthfactor receptor (Hunger, T., et al., 1984, Nature 311:480; Davis, R. J.and Czech, M. P., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:1974). However,this cytoplasmic sequence, which was found in three independent clonesof the tonsillar library, is most likely that of B cell CR1, which isnot phosphorylated after activation of protein kinase C (Changelian, P.S. and Fearon, D. T., 1986, J. Exp. Med. 163:101).

7. EXAMPLE CR1 5′ cDNA Sequences Contain a Fourth Long Homologous Repeat

Analysis of a partial cDNA sequence of CR1 revealed a structure in whichthree LHRs, LHR-B, LHR-C, LHR-D, of 450 amino acids were each comprisedof seven short consensus repeats (SCR) of 65 amino acids characteristicof C3/C4 binding proteins (see Section 6, supra). In the examplesdescribed herein, we describe the cloning and nucleotide sequence of afourth amino-terminal LHR, LHR-A (Klickstein, L. B., et al., 1987,Complement 4:180) by the sequencing of 5′ cDNA clones. Analysis of LHR-Arevealed that it is 99% homologous to LHR-B in the five 3′ SCRs, butonly 61% homologous in the two 5′ SCRs.

7.1. Material and Methods

7.1.1. Construction of a cDNA Library

A selectively primed cDNA library, λHH, was constructed from 3 μg ofpoly (A)⁺ RNA purified from DMSO-induced cells as described (Chirgwin,J. M. et al., 1979, Biochemistry 18:5290; Aviv, H. and Leder, P., 1972,Proc. Natl. Acad. Sci. U.S.A. 69:1408; Ausubel, F. M., et al., 1987,Current Protocols in Molecular Biology, John Wiley & Sons, New York)with the following modifications. LK35.1, a 35-mer oligonucleotide,5′-TGAAGTCATC ACAGGATTTC ACTTCACATG TGGGG-3′, was used in place ofoligo(dT)₁₂₋₁₈ and 40 μCi of α³²P-dCTP were added during second strandsynthesis. One third of the cDNA was cloned in λgt11 and a cDNA librarywas constructed from human tonsilar poly(A)⁺ RNA as described in Section6.1.2, supra. 750,000 independent recombinants were obtained.

7.1.2. Isolation of Clones, Probes, and DNA Sequence Analysis

The probes used for screening cDNA libraries were CR1-1 (Wong, W. W., etal., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7711) (ATCC accession no.57331), CR-2 (Wong, W. W., et al., supra), CR1-4 (Wong, W. W. et al.,1986, J. Exp. Med. 164:1531), and CR1-18, a 252 bp Sau3AI fragment fromthe 0.5 kb EcoRI fragment of cDNA clone λH3.1 corresponding tonucleotides 101-352 in FIG. 1A. Under conditions of high stringency,CR1-18 hybridizes only to cDNA clones encoding either the NH₂-terminalSCR of LHR-A or the signal peptide. The inserts of the cDNA clones weresequenced by the dideoxynucleotide technique (Sanger, F., et: al., 1977,Proc. Natl. Acad. Sci. U.S.A. 74:5463) after subcloning fragments intoM13mp18 and M13mp19 (Yanisch-Perron, C. et al., 1985, Gene 28:351).

7.2. Results

A specifically primed λgt11 cDNA library, λHH, that contained 7.5×10recombinants was prepared with cDNA synthesized from poly (A)⁺ RNA fromDMSO induced HL-60 cells. These cells express only the F allotype of CR1(Lublin, D. M., et al., 1986, J. Biol. Chen. 261:5736) which ispredicted to have four LHRs (Lapata, M. A., et al., 1984,, Nucl. AcidsRes. 12:5707). The primer, LK35.1, was an antisense 35-mer correspondingto nucleotides 896-930 of the partial cDNA sequence of CR1 presented inFIGS. 3A-3D. This oligonucleotide was shown to hybridize to LHR-B, LHR-Cand LHR-D under the conditions of reverse transcription. Two hundred andfifty positive clones were identified in a plating of 3.8×10⁵unamplified recombinant phage screened with a mixture of the CR1 cDNAprobes, CR1-1 and CR1-4. Thirty-eight positive clones were picked andplaque purified. Southern blots of EcoRI-digested DNA from these cloneswere screened with the 23-mer oligonucleotide, KS23.1, 5′-CTGAGCGTACCCAAAGGGAC AAG-3′, corresponding to nucleotides 763-785 of the partialCR1 cDNA sequence of FIG. 3. This probe hybridizes under conditions ofhigh stringency at a single site in the sequence encoding LHR-B but notto sequences encoding LHR-C or LHR-D. The insert of clone λH7.1 (FIG. 9)contained three EcoRI fragments of 1.0 kb, 0.9 kb and 0.4 kb and the twolarger fragments hybridized to KS23.1, indicating that this clonecontained sequences coding for the 3′ {fraction (5/7)} of LHR-A and allof LHR-B. This finding confirmed that LHR-A is highly homologous toLHR-B. Clone λH3.1 (FIG. 9) contained a single KS23.1-positive EcoRIfragment of 1.0 kb and a 5′, 0.5 kb fragment that hybridized weakly withCR1-4 at high stringency. This clone was considered to contain theadditional 5′ sequence completing LHR-A, including SCRs -1 and -2 and0.1 kb of upstream sequence. None of the remaining 36 clones, all ofwhich hybridized with CR1-1, were detected with the probe, CR1-18, a 252bp Sau3AI fragment from the 0.5 kb EcoRI fragment of clone λH3.1 thatdoes not hybridize to sequences encoding LHR-B, -C or -D.

DNA sequence analysis of λH3.1 revealed that the open reading framecontinued to the 5′ end of the cDNA, indicating that the clone did notextend to the translational start site. Therefore, the cDNA libraries,λHH and λS2T, were rescreened with the probe CR1-18 to identify oneclone from each, λH10.3 and λT109.1, respectively. The EcoRI fragmentsof these clones that hybridized with CR1-18 were sequenced as were theinserts from the clones, λH3.1 and λH7. 1. The composite sequence ispresented in FIGS. 1A-1P such that the nucleotide following number 1531in FIG. 1D is nucleotide #1 in FIG. 3A. The overlapping sequences of thecDNA clones from the HL-60 and tonsillar libraries are identical.

Immediately upstream of LHR-A, clones λH10.3 and λT109.1 containidentical putative hydrophobic leader sequences (Von Heijne, G., 1986,Nucl. Acids Res. 14:4683) encoding 41 amino acids, including an ATGmatching the consensus NNA/GNNATGG proposed for eukaryotic translationinitiation sites (FIG. 10) (Kozak, M., 1986, Cell 44:283). A second ATG,located six codons upstream of the chosen ATG and just downstream of anin-frame stop codon, is a poor match for this consensus sequence. Thefirst three amino acids of this leader sequence for CR1, MGA, are thesame as those reported for CR2. The sequences of these two clonesdiverge upstream of the ATG and that from clone λ10.3 is believed torepresent a portion of an intervening sequence, as has been describedfor other CR1 cDNA clones in Section 6, supra.

The signal peptidase cleavage is predicted (Von Heijne, G., 1986, Nucl.Acids Res. 14:4683) to occur between, glycine-46 and glutanine-47,suggesting that the blocked NH₂-terminus of CR1 (Wong., W. W., at al.,1985,.J. Immunol. Methods 82:303; Holeis, V. M., et al., 1986,Complement 3:63) may be due to the presence of a pyrrolidone amide. Thefirst two SCRs of the NH₂-terminal LHR-A contained in these clones areonly 61% identical to the corresponding region of LHR-B, whereas SCRs3-7 of LHR-A are 99% identical to the corresponding SCRs of LHR-B (FIG.10). Comparison of LHR-A with LHR-C reveals that only the third andfourth SCRs of each are highly homologous (99% identical). LHR-A and -Dhave only 68% overall identity, with maximal identity of 81% between thesixth SCR of each LHR. Thus, completion of the 5° cDNA sequence of CR1indicates that the F allotype is comprised of 2039 amino acids includinga 41 amino acid signal peptide, four LHRs of seven SCRs each, twoadditional COOH-terminal SCRs, a 25 residue transmembrane region and ai43 amino acid cytoplasmic domain. There are 25 potential N-linkedglycosylation sites.

7.3. Discussion

The primary structure of the NH₂-terminus and the signal peptide of theF allotype of CR1 has been deduced by the isolation and sequencing of 5′cDNA clones. The highly repetitive nature of the CR1 sequence madecritical the development of an appropriate strategy for the preparationand identification of cDNA clones encoding this region of the receptor.A cDNA library was prepared using as a primer a 35-mer oligonucleotideknown to hybridize under the conditions of reverse transcription toLHR-B, -C and -D, the possibility was considered that this primer mighthybridize also to LHR-A that had been predicted to be highly homologousto LHR-B (see Section 6 supra). Appropriate cDNA clones were identifiedby the use of another oligonuclootide, KS23.1, that hybridizes only toLHR-B under stringent conditions, thereby increasing the probability offinding 5′ cDNA clones. Two clones were found that encompassed almostall of the residual sequence of CR1, and a Sau3AI fragment of one ofthese, CR1-18, had sequence sufficiently unique to permit its use in theidentification of the remaining 5′ clones (FIGS. 9, 10).

A 250 bp probe from the 5′ region of LHR-A, CR1-18, hybridized not onlyto CR1 transcripts of 7.9 and 9.2 kb, but also to a 2 kb transcript inhuman tonsillar RNA under stringent conditions. This cross-hybridizingmRNA was not observed with CR1 cDNA probes from other LHRs or innorthern blots of RNA from dimethyl sulfoxide-induced HL-60 cells andHSB-2 T lymphoblastoid cells. Thus, CR1 contains sequences homologous totwo additional B cell proteins, one that is encoded by this newlyrecognized mRNA, and CR2.

EXAMPLE Expression of Recombinant Human CR1

As described supra, human CR1 cDNA clones have been isolated that span7.0 kb and contain an open. reading frame encoding 2039 amino acids(FIGS. 1A-1P). The proposed precursor form of the receptor includes a 41amino acid signal peptide, four long homologous repeats (LHRs) of 450amino acids with each LHR comprised of 7 short consensus repeats (SCRs),two COOH-terminal SCRs of 65 amino acids, a 25 amino acid transmembranedomain, and a 43 amino acid cytoplasmic region. Thus, the CR1 F allotypecontains 30 SCRs. The NH₂-terminal LHR, LHR-A (see Section 7, supra), is61% identical to the corresponding region of LHR-B in the first two SCRsand 99% identical in the COOH-terminal five SCRs. Restriction fragmentsof eight CR1 cDNA clones were spliced to form a full length construct of6.9 kb and placed downstream of a mouse metallothionein promoter or acytomegalovirus promoter, and transfected into L (mouse) cells or COS(monkey) cells. Recombinant cell surface CR1 was detected by indirectradioimmunoassay and immunofluorescence. No antigen was detected oncells transfected with the parental vector (CR1⁻) only.Immunoprecipitation of transfected, surface ¹²⁵I-labeled, COS (monkey)cells by anti-CR1 monoclonal antibody, and analysis by non-reducingsodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, yieldeda 190,000 daltons molecular weight band which co-migrated with the Fallotype from human erythrocytes. The expression of recombinant CR1antigen of the correct molecular weight (Klickstein, L. B., et al.,1988, FASEB.J. 2:A1833) providers evidence that the cDNA contains theentire coding sequence of human CR1.

8.1. Construction of Plasmid pBSABCD, Containing the Entire CR1 CodingSequence

We describe herein the construction of plasmid pBSABCD, a vectorencoding the full length (SCRs 1-30) CR1 protein.

The 2.3 kb insert from cDNA clone λT8.2 (Klickstein, L. B., et al.,1987, J. Exp. Med. 165:1095; see Section 6, supra) was subcloned intopUC18 as an EcoRI fragment, such that the 5′ end was proximal to theHindIII site in the plasmid polylinker. This plasmid was named p188.2.p188.2 was cut with ApaI and HindIII, and the large 4.7 kb fragmentcontaining CR1 sequence from SCR 26 through the 3′ untranslated regionplus vector sequences was gel-purified.

The insert from cDNA clone λT50.1 (Klickstein, L. B., et al., 1987, J.Exp. Med. 165:1095; see Section 6, supra) was subcloned as an EcoRIfragment into M13mp18. This phage was called 18R50.1. DNA from thereplicative form of this clone was cut with HindIII and ApaI, and the1.45 kb fragment containing CR1 SCRs 18-25 was isolated, ligated to the4.7 kb fragment from p1188.2, and the ligation transformed into E. coliDH5α. This plasmid was called p8.250.1.

The 0.75 kb and the 0.93 kb EcoRI fragments from cDNA clone λT8.3 (Wong,W. W., et al., 1985, Proc. Natl. Acad. Sci. U.S.A 22:7711) weresubcloned into plasmid pBR327. These subclones were called pCR1-1 andpCR1-2, respectively, and contained SCRs 11-14 and SCRs 17-21,respectively. The EcoRI inserts were purified from each. The 0.75 kbpCR1-1 fragment was digested with SmaI, and the digest was ligated topUC18 DNA cut with EcoRI and SmaI. A subclone, p181-1.1, with a 0.5 kbinsert corresponding to SCRs 12-14, was isolated. The 0.93 kb fragmentof pCR1-2 was digested with HindIII, and ligated to pUC19 cut with EcoRIand HindIII, and a subclone, p191-2.1, was isolated that contained a0.27 kb insert containing SCR 17.

The cDNA clone λT6.1 (See Section 6, supra; Klickstein, L. B., et al.,1987, J. Exp. Med. 165:1095; Wong, W. W., et al., 1987, J. Exp. Med.164:1531) was digested with EcoRI, and the 0.37 kb fragmentcorresponding to CR1 SCRs 15 and 16 was subcloned into pBR322. Thisclone was called pCR1-4. Clone p181-1.1 was cut with EcoRI and ScaI, andthe 1.4 kb fragment was isolated. Clone p191-2.1 (Klickstein, L. B., etal., 1987, J. Exp. Med. 165:1095; see Section 6, supra) was digestedwith EcoRI and ScaI and the 2.0 kb fragment was isolated, ligated to the1.4 kb fragment from p181-1.1, and the mixture was transformed into E.coli DH5α. The resulting plasmid was called p1-11-2. Plasmid p1-11-2 wasdigested with EcoRI, and the 0.37 kb insert fragment from pCR1-4 wasinserted by ligation. The resulting plasmid was used to transform E.coli DH5α.

A subclone was chosen that contained a 0.39 kb BamHI-HindIII fragment.This plasmid was called p142 and contained CR1 SCRs 12-17. The 3.5-kbEcoRI-HindIII insert fragment from p8.250.1 was transferred to pGEM3b.This plasmid was called pG8.250.1. The 1.2 HindIII fragment from p142was purified and ligated to pG8.250.1 that had been cut with HindIII. Asubclone was chosen that contained a 2.4 kb PstI-ApaI insert, thusselecting the correct orientation.

This plasmid was called pCD and contained CR1 sequences from SCR 12through the 3′ end.

The cDNA clone λ5′7.1 (Klickstein, L. B., et al., September 1987,Complement 4:180; see Section 7, supra) was cut with PstI, and the 1.35kb fragment corresponding to SCRs 6-12 was isolated and ligated toPstI-cut pCD. The mixture was transformed, and a subclone was selectedwhich contained 1.35 kb and 1.1 kb HindIII fragments. This clone wascalled pBCD.

The cDNA clone λ5′3.1 (Klickstein, L. B., et al., 1987, Complement4:180; see Section 7, supra) was cut with EcoRI, and the digest wasligated to EcoRI-cut pUC18. A subclone, p3.11-1, was isolated, thatcontained a 1.0 kb insert corresponding to SCRs 3-7, which insert wasgel-purified. The cDNA clone λ5′10.3 (Klickstein, L. B., et al., 1987,Complement 4:180; see Section 7, supra) was cut with EcoRI, and the 0.63kb insert containing SCRs 1 and 2 was subcloned into pUC18. This clonewas called p10.3.5. Plasmid p10.3.5 was partially-digested with EcoRI,and a 3.4 kb fragment corresponding to linear plasmid was isolated andligated with the 1 kb fragment from p3.11-1. A subclone, pLA, waspicked, which contained a 1.3 kb PstI fragment, in the correct site ofinsertion and orientation.

The cDNA clone λT109.4 (Klickstein, L. B., et al., 1987, Complement4:180; see Section 7, supra) was digested with EcoRI, and subcloned intopUC18. A subclone was chosen that contained a 0.55 kb EcoRI fragmentcorresponding to the 5′ untranslated region through the leader sequenceand SCRs 1 and 2. The plasmid p109.4 was cut with PstI and BspMII, and a3.0 kb fragment containing the vector, leader sequence, and SCR 1, wasisolated. The fragment was ligated to a 0.81 kb PstI-BspMII fragmentfrom pLA that contained SCRs 2-5. This new plasmid was called pNLA. Theplasmid pNLA was partially digested with EcoRI and completely digestedwith PstI, and a 1.1 kb EcoRI-PstI fragment containing CR1 sequence fromthe leader sequence through SCR 5 was isolated and ligated topBluescript KS+ (Stratagene, San Diego, Calif.) to put an XhoI site onthe 5′ side of the cDNA. This plasmid was called pXLA.

The plasmid pBCD was cut with EcoRV and then partially digested-withPstI, and a 6.0 kb PstI-EcoRV fragment containing CR1 sequence from SCR6 through the 3′ untranslated region was isolated and ligated toPstI+SmaI-digested pXLA. The resulting bacterial expression plasmid,which contains the entire CR1 cDNA coding sequence, was called pBSABCD.

8.2. Construction and Assay of Plasmid piABCD, A Mammalian ExpressionVector Containing The Entire CR1 Coding Sequence

The pBSABCD plasmid was digested with XhoI and NotI, and the insert wasligated downstream from the CMV promotor in the 4.4.kb fragment of theexpression vector, CDM8 (Seed, B., 1987, Nature 329:840-842), which alsohad been cut with these restriction enzymes. The resulting constructionwas termed piABCD (FIG. 11B). Alternatively, the 6.9 kb XhoI-NotIfragment was ligated downstream from the metallothionein promoter in theexpression vector, pMT.neol, which had also been cut with theserestriction enzymes. The resulting construction was termed pMTABCD (FIG.11A).

Sheep erythrocytes sensitized with rabbit antibody (EA) and limitedamounts of C4b [EAC4b(lim)] and 12,000 cpm ¹²⁵I-C3b per cell[EAcC4b(lim),3b] were prepared by sequential treatment of EAC4b(lim)(Diamedix) with C1, C2. and ¹²⁵I-C3 followed by incubation for 60Minutes at 37° C. in gelatin veronal-buffered saline containing 40 mMEDTA. Alternatively, methylamine-treated C3 [C3(ma)] were covalentlyattached to sheep E (erythrocytes) treated with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (Sigma) (Lambris, J. D., etal., 1983, J. Immunol. Methods 65:277). EAC4b were prepared withpurified C4 (Hammer, C. H., et al., 1981, J. Biol. Chem. 256:3995).

Both piABCD and pMTABCD were transfected by the DEAE(diethylaminoethyl)-dextran method into COS (monkey) cells. RecombinantCR1 was detected on the surface of the transfected cells byimmunofluorescence using the anti-CR1 monoclonal antibody, YZ-1; and byimmunoprecipitation of ¹²⁵I-labeled cells followed by non-reducingSDS-PAGE, which revealed a protein having a mobility identical to thatof CR1 immunoprecipitated from human erythrocytes of a donor homozygousfor the F allotype (Wong, W. W., et al., 1983, Jr. Clin. Invest.72:685); and by formation of rosettes with sheep erythrocytes coatedwith C3b (Fearon, D. T., 1980, J. Exp. Ned. 152:20). The identicalelectrophoretic mobilities of the native and recombinant CR1 proteinsconfirmed that the CR1 F allotype contains SCRs 1-30.

In addition, murine L cells were co-transfected. by the DEAE-dextranmethod (Ausubel, F. M., et al., 1987, Current Protocols-in MolecularBiology, Seidman, J. G. and Struhl, K., eds., John Wiley & Sons, NewYork; Seed, B., 1987, Nature 329:840) in duplicate with 0, 2, or 4 μg ofeither piABCD or pMTABCD and 2 μg of pXGH5, a reporter plasmid thatdirects the expression of growth hormone (Selden, R. F., et al., 1986,Mol. Cell. Biol. 6:3173). The cells were harvested after two days andassayed for expression of CR1 by binding of YZ1 monoclonal anti-CR1antibody. There was a dose response relationship between recombinantplasmid DNA and the expression of CR1 antigen (Table II).

TABLE II DOSE RESPONSE OF RECOMBINANT CR1 AND HUMAN GROWTH HORMONE INCO-TRANSFECTED L CELLS YZ1 Anti- CR1 mAB Growth Plate pXGH5 pMTABCDpIABCD RIA* Hormone Number (μg) (μg) (μg) (cpm) (ng/ml) 1 2 0 0 1444 1202 2 0 2 6058 130 3 2 0 2 6531 140 4 2 0 4 10620 180 5 2 0 4 9898 80 6 22 0 3111 180 7 2 2 0 2747 160 8 2 4 0 3547 160 9 2 4 0 3337 140 *Forradioimmunoassay (RIA), replicate samples of 3 × 10⁵ transfected cellsin 0.1 ml phosphate-buffered saline containing 1% bovine serum albuminand 0.02% sodium azide were incubated at 0° C. for 60 minutes with 3μg/ml YZ-1 IgGl anti-CR1 (Changelian, P.S., et al., 1985, J. Immunol.134:1851). The cells were washed and resuspended in 0.1 ml of buffercontaining 1-2 μCi/ml of ¹²⁵I-F(ab′)₂ goat-anti-mouse IgG or¹²⁵I-protein A. # After 1-2 hours 0° C., the cells were washed andassayed for ¹²⁵I.

The plasmid, piABCD, directed the expression of nearly three-fold moreCR1 antigen than did pMTABCD. The growth hormone concentration in theculture medium varied by less than two-fold with the exception of plate5. Additional experiments revealed that piABCD directed the transientexpression of three-fold more CR1 antigen in COS cells than in L cells.

CR1 antigen was present in clusters on the surface of the transfectedCOS calls when assessed by indirect immunofluorescence of calls stainedwith YZ1 anti-CR1 mAB FIGS. 12a-12 d. This distribution of recombinantCR1 on COS cells resembles that of wild type CR1 on human leukocytes(Fearon et al., 1981, J. Exp. Med. 153:1615).

The molecular weight of the recombinant CR1 was determined by surfaceiodination of COS cells transfected with piABCD, immunoprecipitation ofcell lysates with Sepharose-YZ1, SDS-PAGE and autoradiography. Therecombinant CR1 had a molecular weight of 190,000 unreduced which isequivalent to that of the F allotype and less than that of the Sallotype of erythrocyte CR1 (FIG. 14).

The C3b-binding and C4b-binding function of recombinant CR1 was assayedby the formation of rosettes between the transfected COS calls and EAC4bor EAC4b(lim),3b. In 31 separate transfections, 5%-50% of COS cellstransfected with the plasmid, piABCD, bound five or more EAC4b orEAC4b(lim),3b FIGS. 13a-13 d. The COS cells expressing CR1 did not formrosettes with EAC4b(lim),3bi, although this intermediate did formrosettes with Raji B lymphoblastoid cells expressing CR2.

8.3. Expression of CR1 Fragments

Expression vectors encoding part of the CR1 coding sequence (deletionmutants) were constructed as described infra, and found to express theirrespective CR1 inserts when transformed into COS cells. The CR1fragments were expressed as cell-surface proteins.

8.3.1. Construction of Deletion Mutants piBCD, piABD, piACD, piAD, piBD,piCD and piD

The construction of these deletion mutants was performed by takingadvantage of the presence of a single BsmI site in a homologous positionnear-the amino-terminus of each of the four CR1 long homologous repeats(LHRs), and the absence of BsmI sites elsewhere in the CR1 cDNA andBluescript vector (Stratagene, San Diego, Calif.).

Ten micrograms of the plasmid pBSABCD were partially digested with 50units of the restriction enzyme BsmI for 45 minutes, and the digest wasfractionated by agarose gel electrophoresis. DNA fragments of 8.55 kb,7.20 kb and 5.85 kb were purified that corresponded to linear segmentsof the parent plasmid that lacked one, two or three LHRs, respectively.Each of the three fragments was ligated to itself and the ligations usedseparately to transform competent E. coli DH5α to ampicillin resistance.

The 8.55 kb fragment was generated as the consequence of cleavage ofpBSABCD at two adjacent BsmI sites, thus there are three possibleproduct plasmids after ligation, pBCD, pACD or pABD, where the capitalletters represent the LHRs that remain in the plasmid. These weredistinguishable by restriction mapping with SmaI. DNA was prepared from12 colonies, digested with SmaI, and separated by agarose gelelectrophoresis. Five clones had two SmaI fragments of 2.5 kb and 6.1kb, corresponding to deletion of the coding sequence of LHR-A, thusrepresenting pBCD. Three clones had a single linear fragment of 8.5 kbcorresponding to pACD. Four clones had two SmaI fragments of 1.2 kb and7.4 kb, which was expected for the deletion of the coding sequence ofLHR-C, producing pABD. The 5.6 kb insert of each of these threeconstructions was gel-purified after double digestion with XhoI andNotI, and ligated to the expression vector CDM8 that had beengel-purified after digestion with the same restriction enzymes. E. coliDK1/P3 was transformed with the ligation mixtures and DNA was preparedfrom five colonies of each. The presence of the deleted CR1 cDNA insertin the expression vector was shown in each case by SacI digestion, whichrevealed the expected two fragments of 4.20 kb and 5.75 kb. Theseplasmids were called piBCb, piACD and piABD.

The 7.20 kb fragment from the partial digestion of pBSABCD was aconsequence of BsmI digestion at three adjacent sites or, equivalentlywith respect to the large fragment, at two sites with a single uncutsite between them, thus there were two possible products obtainableafter transformation, pAD and pCD. These were distinguished by doubledigestion with XhoI and PstI, which yielded two fragments of 1.0 kb and6.2 kb in the case of pAD, and a linear fragment of 7.2 kb for pCD. The4.2 kb insert from each of these plasmids was gel-purified after doubledigestion with XhoI and NotI, and subcloned into CDM8 as above. Thepresence of the deleted CR1 cDNA in the expression vector was shown bydouble digestion with PstI and Bg1II. The clone piAD had fragments of2.4 kb and 6.2 kb, while piCD had a single fragment of 8.6 kb.

The 5.85 kb fragment from the BsmI digestion of pBSABCD represents aproduct of complete digestion and a single clone, pD, was obtained aftertransformation of E. coli DH5α. This was confirmed by double digestionwith HindIII and Bg1II which yielded the expected 3.7 kb and 2.2 kbfragments. The 2.9 kb insert of the clone was gel-purified after doubledigestion with XhoI and NotI and ligated to the expression vector asabove. HindIII digestion of the resulting piD clone yielded the expected7.3 kb fragment, a XhoI+Bg1II double digest gave 2.2 kb and 5.1 kbfragments, and a SacI digest resulted in the expected 1.5 kb and 5.8 kbfragments.

The plasmid pBD was prepared by BsmI partial digestion of pBCD. Thelinear 7.2 kb fragment corresponding to cleavage of two adjacent BSmIsites was gel-purified, self-ligated as above, and E. coli DH5α wastransformed to ampicillin resistance. pBD was identified by the presenceof 1.2 kb and 6.0 kb fragments upon SmaI digestion. The 4.2 kb insertwas purified after double digestion with XhoI and NotI, and transferredto CDM8 as above. The clone piBD was confirmed by observation of theexpected 0.8 kb and 7.8 kb fragments after HindIII digestion.

COS cells transiently expressing the piABCD, piBCD, piCD, and piDconstructs,,respectively, were surface-labelled with ¹²⁵I, andimmunoprecipitated with anti-CR1 antibody. On SDS-PAGE followingreduction, the product of the piABCD construct comigrated with the Fallotype of CR1, while the deletion mutants demonstrated stepwisedecrements of approximately 45,000 daltons, indicative of the deletionof one, two and three LHRs, respectively (FIG. 17).

8.3.2. Construction of Deletion Mutants piP1, piE1, piE2, piE-2, piU1,piU-2 and piA/D

The plasmid piABCD was completely digested with BstEII and the twofragments at 1.35 kb (a doublet) and 8.6 kb were gel-purified, mixed,and ligated, and E. coli DK1/P3 was transformed to ampicillin andtetracycline resistance. Colonies were screened by hybridization withthe CR1 cDNA probe CR1-4 (see Section 8.1, supra), and strongly positiveclones were picked and further screened by digestion with SmaI. piE1 wasidentified by the presence of two fragments at 2.7 kb and 7.3 kb, andpiE2 was identified by a single 10.0 kb linear fragment. piE-2 wasidentified as a weakly CR1-4 positive clone that contained a single 8.6kb SmaI fragment.

The plasmid piP1 was obtained by complete digestion of piABCD with PstIand gel-purification of the large, 10.0 kb fragment. This fragment wasligated and E. coli DK1/P3 was transformed with the mixture. Theresulting plasmid, piP1, contained a single, 10.0 kb SmaI fragment.

The plasmids piU1 and piU-2 were prepared by first transforming thedcm⁻strain GM271/P3 with the plasmid pXLA, and isolating DNA. This DNAwas double digested with StuI and NotI, and the 3.3 kb fragment wasgel-purified. The plasmid pBSABCD was partially digested with NsiI, andthe resulting four base pair 3′ overhangs were removed by treatment withthe Klenow fragment of E. coli DNA polymerase I. The DNA was thendigested to completion with NotI, and fragments of 5.4 kb and 4.0 kbwere gel-purified. These were ligated to the 3.3 kb StuI-NotI fragmentfrom pXLA, and the ligation mixture was used to transform E. coli DH5αto ampicillin resistance. Colonies were screened by hybridization to theCR1 cDNA probe CR1-4, and positive clones were further checked byrestriction digestion with HindIII which yielded three fragments of 0.8kb, 1.3 kb and 6.5 kb for pU1, and two fragments of 0.8 kb and 6.5 kbfor pU-2. The StuI-blunted NsiI splice was confirmed to be in-frame byDNA sequencing of these plasmids. The inserts of pU1 and pU-2, 5.6 kband 4.2 kb, respectively, were gel-purified after XhoI and NotI doubledigestion, and were ligated to the expression vector CDM8 as describedsupra. The structures of the clones, piU1 and piU-2, were confirmed byrestriction digestion with XhoI+PstI, yielding the expected twofragments of 1.2 kb and 8.8 kb for piU1 and a linear 8.7 kb fragment forpiU-2.

The plasmid piA/D was prepared by first digesting piABCD with PstI tocompletion. The PstI digest was then partially digested with ApaI, andthe 3′ overhangs were removed with the Klenow fragment of E. coli DNApolymerase I. The DNA was then fractionated by agarose gelelectrophoresis and the 7.5 kb fragment was isolated, ligated, and usedto transform E. coli DK1/P3 to ampicillin and tetracycline resistance.The construction was confirmed by double digestion with KpnI+SacI, whichyielded the expected four fragments of 0.8 kb, 1.5 kb, 1.7 kb and 3.3kb.

9. EXAMPLE Identification of C3b and C4b Binding Domains 9.1. Assays andResults

Plasmids piABCD, piAD, piCD, and piD, containing the LHR(s) denoted bythe capital letter(s) of their names, were transformed into COS cells,which were used in assays to assess the ability of their encoded CR1fragments to bind C3b or C4b. Binding assays were carried out byobservation of erythrocyte rosetting resulting from the binding of C3bor C4b-coated red cells by COS cells expressing a full-length CR1molecule or a CR1 deletion mutant on their cell surface (transientexpression). Transfected cells, 1-4×10⁶/ml, were incubated with C3- orC4-bearing erythrocytes, 2-6×10⁸/ml, in 0.02 ml for 60 minutes at 20° C.The percentage of transfected cells forming rosettes was evaluatedmicroscopically with a transfected-cell scored as a rosette if therewere at least five adherent erythrocytes.

The results are shown in Table III.

TABLE III FORMATION OF ROSETTES BETWEEN COS CELL TRANSFECTANTSEXPRESSING RECOMBINANT FORMS OF CR1 AND SHEEP ERYTHROCYTES BEARINGC3(ma) OR C4(ma) % Transfectants Forming Rosettes % TransfectantsFluorescent with Anti-CR1 COS Cell Transfectant EC3 (ma)* EC4 (ma)^(#)piABCD 109 (3)^(π) 62 (2) piAD 8 ^(π) 107 ^(π) piBD 107 ^(π) 12 ^(π)piCD 127 ^(π) 32 ^(π) piD 0 ^(π) 0 ^(π) piA/D 11 (2) 83 (2) piE-2 1 (1)102 (1) *The numbers of C3(ma) per erythrocyte were 60,000, 350,000 and900,000, respectively, in the three experiments using this intermediate.^(#)The number of C4(ma) per erythrocyte were 160,000 and 140,000,respectively, in the two experiments using this intermediate. ^(π)Numberof experiments.

In each of three separate experiments, the proportion of COS cellsexpressing the full length piABCD construct that formed rosettes withthe EC3(ma) was similar to the fraction having detectable recombinantreceptor, as assessed by immunofluorescence using either YZ1 monoclonalanti-CR1 antibody or rabbit anti-CR1 antiserum (Table III). In contrast,cells expressing piD did not form rosettes, indicating that a C3-bindingsite(s) lust reside in or require the presence of LHR-A, -B or -C. Asite was shown to be present in both LHR-B and -C by demonstrating thatcells expressing either the piBD or piCD constructs formed rosettes withEC3(ma). Calls expressing piAD, piA/D, or piE-2 did not have equivalentC3-binding function. As the piE-2 construct differs from piCD only inhaving SCR-1 and -2 of LHR-A instead of the first two SCRs of LHR-C, thefunction of the C3-binding site in LHR-C must require these NH₂-terminal SCRS.

The proportion of COS cells expressing the full length piABCDrecombinant that formed rosettes with EC4(ma) was less than the fractionrosetting with EC3(ma), perhaps reflecting fewer C4(ma) per erythrocyte(Table III) or fewer C4-binding sites per receptor. Deletion mutantshaving all or part of LHR-A, the piAD, piA/D and piE-2 constructs, boundEC4(ma) better than did the deletion mutants, piBD and piCD; piD lackedthis function. Thus, the C4-binding site of CR1 resides primarily inLHR-A, although secondary sites may be present in LHR-B and -C. Theimproved rosetting capability of the piE-2 construct relative to that ofpiCD suggests that SCR-1 and -2 of LHR-A are involved in the C4 bindingsite.

Radioimmunoassay of the binding of YZ1 monoclonal anti-CR1 antibodyindicated significant uptake by COS cells expressing the piABCD, piAD,piBD, and piCD constructs (Table IV). Cells transfected with piD orpiA/D, which is comprised of the five NH₂-terminal SCRs of LHR-A and thethree COOH-terminal SCRs of LHR-D, did not bind YZ1 anti-CR1 antibody,although-the products of these constructs bound polyclonal anti-CR1antiserum (Table IV). Thus, the YZ1 epitope is repeated in LHR-A, -B and-C, is not present in the NH₂-terminal SCRs of LHR-A, and is not presentor is inaccessible in LHR-D.

TABLE IV BINDING OF MONOCLONAL AND POLYCLONAL ANTI-CR1 ANTIBODY TO COSCELL TRANSFECTANTS EXPRESSING RECOMBINANT FORMS OF CR1 Bound Bound COSCell YZ1 Monoclonal Rabbit Polyclonal Transfectant Antibody* Antibody*piABCD 2362 12277 piAD 2879 19891 piBD 3646 21922 piCD 2189 19926 piA/D410 23052 piD 404 16386 CDM8 428 4886 *Replicate samples of 3 × 10⁵transfected cells in 0.1 ml phosphate-buffered saline containing 1%bovine serum albumin and 0.02% sodium azide were incubated at 0° C. for60 minutes with 3 μg/ml YZ1 IgGl anti-CR1 mAb (Changelian, P.S., et al.,1985, J. Immunol. 134:1851) or with 90 μg/ml rabbit IgG anti-CR1antibody. The cells were washed and resuspended in 0.1 ml of buffercontaining 1-2 μCi/ml of ¹²⁵I-F(ab′)₂ # goat-anti-mouse IgG or¹²⁵I-protein A. After 1-2 hours at 0° C., the cells were washed andassayed for ¹²⁵I. Values shown are the mean of duplicate determinations,cpm per 3 × 10⁵ COS cells.

9.2. Discussion

The conserved BsmI site found midway through the coding sequence of thefirst SCR in each LHR permitted the construction of a series of deletionmutants that corresponded closely to the boundaries of the LHRs, andmaintained the open reading frame and the appropriate positions of thefour cysteines necessary for the presumed disulfide bond formation (FIG.16). Comparison of the C3(ma)- and C4(ma)-binding functions of thesedeletion mutants distinguished not only the LHRs having thesespecificities, but also those SCRs critical for determining the ligandspecificity. Thus, the capacity of piAD, piA/D, and piE-2 forms of thereceptor, but not the piD form, to mediate rosette formation between thetransfected COS cells and EC4(ma) indicated that the NH₂-terminal twoSCRs or LHR-A contained a site for interaction with this complementprotein (Table III). This site was only relatively specific for C4(ma)because transfectants expressing piAD and piA/D also were capable ofbinding EC3(ma) (Table III). The C3(ma)-binding function of thereceptors encoded by the piBD and piCD constructs, demonstrated byrosette assay and factor I-cofactor function for cleavage of C3(ma)(Table III; FIG. 18), indicated the presence of sites specific forC3(ma) in the first two SCRs of these LHRs. These sites also werecapable of interacting with C4(ma) (Table III). Thus, there arepreferential, but overlapping, C4- and C3-binding activities in LHR-A,-B and -C.

Alternatively, the capacity of the COS cells expressing the piBD andpiCD constructs to bind EC4(ma) may have been caused by the transfer ofnucleotides encoding the NH₂-terminal 36 amino acids from SCR-1 of LHR-Ato LHR-B and -C through the ligation of the BsmI fragments. However,these 36 amino acids alone did not confer on the piD productC4-rosetting function. We cannot exclude a secondary function of LHR-Din these reactions because this LHR was; present in all the constructsassayed for function. The finding of three distinct ligand recognitionsites in CR1, two for C3b and one for C4b (FIG. 19), indicates that eachreceptor molecule may be capable of effectively binding complexesbearing multiple C4b and C3b molecules despite having a relatively lowaffinity for monovalent ligands (Arnaout, M. A., at al., 1983,.Immunology 48:229). This finding also provides an explanation for theinability of soluble C4b to inhibit formation of rosettes betweenerythrocytes bearing C3b and a human B lymphoblastoid cell line(Gaither, T. A., et al., 1983, J. Immunol. 131:899). Possible ligandsfor which CR1 would be especially adapted may be the molecularcomplexes, C4b/C3b and C3b/C3b, that are generated during activation ofthe classical and alternative pathways, respectively. Since there aredistinct binding sites in three of the four LHRs, the CR1 structuralallotypes differing by their number of LHRs may have significantfunctional differences caused by variations in the number of ligandbinding sites. Although in vitro studies have not reported differingbinding activities of the F, S and F′ (A, B and C, respectively)allotypes, the smaller F′ allotype presumably having only three LHRsmight have an impaired capability to clear immune complexes. The F′allotype has been reported possibly to be associated with systemic lupuserythematosus (van Dyne, S., et al., 1987, Clin. Exp. Immunol. 68:570).

10. EXAMPLE Demonstration of Factor I Cofactor Activity

The recombinant CR1 protein, and specific fragments thereof, in bothcell-surface and solubilized forms, were demonstrated to have C3b factorI cofactor activity.

Assays of factor I cofactor activity were carried out by modificationsof a published procedure (Fearon, D. T., 1979, Proc. Natl. Acad. Sci.U.S.A. 76:5867).

For assay of factor I cofactor activity of solubilized CR1 andfragments, cell-surface CR1 protein and fragments were solubilized withNonidet P-40, and the lysate was immunoprecipitated with anti-CR1monoclonal antibody YZ-1 coupled to Sepharose beads. Detergent lysatesof 1×10⁶ transfected COS cells were immunoprecipitated sequentially withSepharose UPC10 anti-levan and Sepharose-YZ-1. The immunoprecipitate wasthen assayed for factor I cofactor activity by incubation of the washedbeads for 60 minutes at 37° C. with 0.5 μg of ¹²⁵I-C3(ma) and 200 ng offactor I in 0.05 ml PBS, 0.5% NP-40. After incubation, the supernatantcontaining radiolabeled C3(ma) was analyzed by SDS-polyacrylamide gelelectrophoresis and autoradiography. Factor I cofactor activity wasindicated by the appearance on the autoradiogram of lower molecularweight forms of the alpha chain of C3(ma) resulting from proteolyticcleavage by factor I.

For assay of factor I cofactor activity of cell-surface CR1 andfragments, transfected COS cells carrying a CR1 expression vector(piABCD, piAD, piBD, piCD, or piD, described supra) were incubated with0.5 μg ¹²⁵I-C3(ma) and 0.2 μg factor I (Fearon, D. T., 1977, J. Immunol.119:1248), and analyzed as described supra.

The factor I-cofactor activity of cell-surface. recombinant CR1 is shownin FIG. 15. Factor I cleaved the alpha chain of C3(ma) into fragments ofmolecular weights. 76,000 and 46,000 only in the presence ofimmunoimmobilized, recombinant CR1 or factor H (FIG. 15). The regionscorresponding to bands from the autoradiogram were excised from the geland assayed for ¹²⁵I to determine the amount of alpha chain cleaved. Inthe presence of factor H, 91% of the alpha chain was cleaved while inthe presence of increasing amounts of recombinant CR1, 26%, 41%, and55%, respectively, was cleaved. Although the COS cells transfected withthe CDM8 vector alone contained some endogenous factor I-cofactoractivity, an increase in this function was evident with COS cellstransfected with piABCD, piBD and piCD (FIG. 18). No enhanced cleavageof ¹²⁵I-C3(ma) was seen with COS cells transfected with piAD or piD.Thus, among these constructs, only the deletion mutants, piBD and piCD,that conferred on COS cells a capacity for binding C3, also had factorI-cofactor activity for cleavage of C3.

The results of the assays for factor I cofactor activity with bothcell-surface and solubilized forms of CR1 and fragments thereof areshown in Table V.

TABLE V FACTOR I COFACTOR ACTIVITY OF CELL-SURFACE AND SOLUBILIZED FORMSOF CR1 and CR1 FRAGMENTS Factor I Cofactor Activity^(b) Plasmid^(a)Cell-Surface Solubilized piABCD + + piAD − − piBD + ND^(c) piCD + + piD− ND^(d) ^(a)Encoding the assayed CR1 protein or fragment, andtransfected into COS cells for expression. ^(b)(+) denotes an increasein cofactor activity above the endogenous level observed upontransfection with the CDM8 vector alone. ^(c)Not determined. ^(d)Notdetermined, due to the absence from LHR-D of the epitope recognized byanti-CR1 monoclonal antibody YZ-1.

As shown in Table V, expression of piABCD (encoding a full-length CR1protein), piBD (encoding LHR-B and -D) or piCD (encoding LHR-C and -D)produced a CR1 product with C3b factor I cofactor activity. The data ofTable V thus provide evidence that the CR1 protein or a fragment thereofcan promote complement inactivation.

11. EXAMPLE Expression of Recombinant Soluble CR1

The CR1 cDNA was modified by recombinant DNA procedures so that asoluble form (sCR1) of CR1 or CR1 fragments was produced. The sCR1constructs were expressed in a mammalian system where the expressedprotein was secreted from the cells. Large quantities of the solublepolypeptides were produced, which, in contrast to the membrane boundform of CR1 proteins, did not have to be solubilized to obtain them insolution.

11.1. Materials and Methods

11.1.1. Enzyme Digestions

All restriction enzyme digestions, linker ligations, and T4 DNA ligaseand E. coli DNA polymerase reactions were done according to themanufacturer's (New England Biolabs, Inc., Beverley, Mass.)recommendations. E. coli DH1 or DH5α were made competent by theprocedure of Morrison, D.A., 1979, Meth. Enzymol 68:326-331. Competentbacterial cells were transformed with DNA according to Maniatis, T., etal., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, New York. Plasmids were purified byalkaline lysis or by the boiling method (Maniatis, T., et al., supra).

11.1.2. DNA Fragment Isolations

DNA fragments were purified from agarose (BioRad, Richmond, Calif.) gelsas follows. The appropriate DNA band was excised from the gel using ablade, and the agarose slice was placed on a piece of parafilm, slicedinto very small pieces, and transferred to a new piece of parafilm. Theagarose pieces were crushed, and the agarose transferred to a 1.5 mltube. An equal volume of phenol (Ultra pure, BRL, Gaithersburg, Md.) wasadded, the mixture vortexed, then frozen at −70° C. for 10 minutes, andcentrifuged for 10 minutes. The aqueous phase was further extractedtwice with phenol/chloroform (1:1), and twice with chloroform. The DNAwas then ethanol precipitated, the pellet washed, dried in vacuo, andresuspended in 10 mM Tris-HC1, pH 7.0, 1 mM EDTA.

DNA fragments were isolated from low gelling temperature agarose (FMC,Corp., Rockland, Me.) As follows. The appropriate DNA band was excisedfrom the agarose gel, placed in a 1.5 ml tube, and melted a 65° C. for15 minutes. The liquified gel was extracted with phenol containing 0.1%sodium dodecyl sulfate (SDS, ultra pure, BRL, Gaithersburg, Md.). Theaqueous phase was further extracted once with phenol-SDS and twice withchloroform. The DNA was then ethanol precipitated in 2.0 M NH₄Acetate,dried, and resuspended in water.

11.1.3. Transfection into Mammalian Cells

Transfection of DNAs into mammalian cells was performed by the CaPO₄precipitation and glycerol shock procedure of Graham and van der Eb(1973, Virology 52:456-467). DUX B11 CHO cells, after being incubatedwith the DNA-calcium phosphate preparation for 4 to 6 hours, weresubjected to glycerol shock by removing the growth medium by aspirationand adding 5 ml of 20% glycerol DMEM medium for 1 minute. Cells werethen washed twice in complete alpha MEM and incubated in this medium for48 hours.

11.1.4. CHO Transfectant Cell Culture

DUX B11 CHO cell transfectants were grown in DHFR (dihydrofolatereductase) selection medium consisting of alpha MEM medium (Gibco)without nucleosides, supplemented with 10% dialyzed fetal calf serum(Gibco) and 4 mM L-glutamine. Amplification was carried out by growingcells in increasing concentrations of methotrexate (Sigma, #A-6770,Amethopterin). (Kaufman, R. J., et al., 1985, Molec. Cell Biol.5:1750-1759).

11.1.5. ELISA for the Detection of Soluble CR1 Levels

11.1.5.1. CR1 Standards

Detergent lysates of hemoglobin-free red blood cell (RBC) ghosts wereused as a CR1 standard in the ELISA (enzyme-linked immunosorbent assay).The ghosts were prepared as previously described (Wong, W. W. and FearonD. T., 1987, Meth. Enzymol 150:579-585). Briefly, expired whole bloodwas obtained from the Red Cross. The red cells were washed three timesin PBS, then lysed in 6 volumes of hypotonic lysis buffer (10 mM Tris pH8, 0.1 mM PMSF (phenyl methyl sulfonyl fluoride), 0.1 mM TPCK(tosylamide-phenylethyl chloromethyl ketone), aprotonin, 2 mM EDTA). Theghosts were washed several times in lysis buffer, counted in ahemocytometer, aliquoted and frozen at −70° C. until needed. For the CR1ELISA, ghosts were diluted to 1.6×10⁸ ghosts/ml in solubilizing buffer(10 mM Tris pH 8, 50 mM KC1, 0.2% NP40, 0.3% DOC, 6.2 mM PMSF, 0.2 mMiodacetamide, aprotonin, 0.1 mM TPCK, 2 M EDTA, 0.2% NaN3) and seriallydiluted to 2.5×10⁶ ghosts/ml for use as standards in the ELISA.Absorbances at 490 nm were plotted and any unknown sample run wasreferred to the plot to obtain ghost equivalents/ml.

11.1.5.2. CR1 ELISA

Immulon-II plates were coated with 100 μl/well of a 0.4 μg/mlconcentration of an anti-CR1 monoclonal antibody (clone J3D3, AMAC IOT17) (Cook, J., et al., 1985, Molec. Imunol. 22:531-538) in PBS andincubated overnight at 4° C. The antibody solution was then discardedand the plates were blocked by the addition of blocking buffer (1.0% BSAin PBS) at 300 μl/well and incubation at 37° C. for 2 hours. Afterblocking, plates were used immediately or stored at 4° C. until needed.

Plates were washed three times using PBS containing 0.05% Tween-20.Samples were added at 100 μl/well in duplicate and incubated 2 hours at37° C. If necessary, samples were diluted in solubilizing buffer.Standard RBC ghosts were included on each plate. After sampleincubation, plates were washed three times and a conjugate (Wilson, M.B. and NaKane, P. K., 1978, Immunofluorescence and Related StainingTechniques, North Holland Biomedical Press, pp. 215-224) of horseradishperoxidase (HRP) and the monoclonal antibody YZ1 (Changelian, P. S., etal., 1985, J. Immunol 184:1851-1858) was diluted 1:8000 in 50% FCS, 50%blocking buffer and added at 100 μl/well. After incubating for two hoursat 37° C., the plates were again washed three times with PBS containing0.05% Tween-20. The substrate orthophenylenediamine (OPD) was added at0.2% concentration in substrate buffer (0.36% citric acid H₂O, 1.74%Na₂HPO₄ .7H₂O, 0.1% thimerosal, 0.4% H₂O₂, pH 6.3) at 100 μl/well. Thereaction was stopped after 20 minutes at room temperature using 50μl/well of 2 N H₂SO₄. Absorbances at 490 nm were read.

11.2. Genetic Modifications of CR1 Coding Sequences

CR1 cDNA is composed of approximately 6,951 nucleotide base pairs (FIGS.1A-1P, Sections 6, 7, supra). The translational stop signal of thenative cDNA is located at base pair 6145. The protein is amembrane-bound receptor molecule composed of four long homologousrepeats (LHRs) which are exposed on the exterior surface of the cellmembrane, plus a membrane-spanning domain of approximately 25 aminoacids, followed by a carboxyl terminal region extending into thecytoplasm. This cytoplasmic domain consists of forty-three amino acids.The strategy we used to produce soluble CR1 molecules (sCR1) was toremove the transmembrane region that anchors a protein in the cellmembrane and then to express the truncated constructs as secretedpolypeptides.

11.2.1. Construction of pBSCR1c

Plasmid pBSABCD (Example 8, supra) contains the CR1 CDNA fromnucleotides 1 to 6860 and lacks the untranslated sequences 3′ to theEcoRV site at nucleotide 6860. CR1 cDNA possesses a unique BalIrestriction endonuclease recognition site at base pair 5914, twenty-ninebase pairs away from the start of the transmembrane domain. pBSABCD wasfirst digested with BalI to produce a linear molecule with flush endsand was then ligated using T4 DNA ligase to a synthetic oligonucleotideconsisting of two 38 nucleotide complementary strands with the followingsequence:

5′: CCAAATGTACCTCTCGTGCACATGATGCTtaaCTCGAG

3′: GGTTTACATGGAGAGCACGTGTACTACGAATTGAGCTC

The resulting molecule had a restored BalI site and an altered sequencewhich reproduced the native CR1 sequence up to and including the alanineresidue at the start of the transmembrane domain. In addition, atranslational stop signal (in lower case and underlined above) had beenintroduced immediately after the alanine, followed by an XhoIrestriction site to faciliate subcloning the altered cDNA.

XhoI digestion of this plasmid (designated pBSCR1c) excised the cDNAinsert (designated sCR1c) by cutting at the oligonucleotide-added XhoIsite in the cDNA and at the XhoI site in the pBSKS+® multiple cloningsite at the 5′ end of the CR1 cDNA. pBSCR1c contains the followingC-terminal sequences:

Base No. 5911: CTGGCCAAATGTACCTCTCGTGCACATGATGCTTAACTCGAG Amino Acids: L  A  K  C  T  S  R  A  H  D  A  END XhoI                                      site

11.2.2. Construction of pBSCR1s

A second sCR1 construct lacking a transmembrane region was generated asfollows. pBSABCD was digested with SacI which cut at the unique SacIsite at nucleotide base pair 5485 in the CR1 cDNA and at the SacI sitein the multiple cloning site of the-host plasmid, located at the 3′ endof the CR1 cDNA. This digestion resulted in the excision of 1375nucleotides of DNA sequence from the 3′ end of the cDNA. This fragmentwas then removed electrophoretically. The exposed ends of the resultingplasmid, containing the remaining sCR1 cDNA, were made flush using T4DNA polymerase and a blunt-end ligation was performed. The Pharmaciauniveral translation terminator (catalog #27-4890-01, Pharmacia, Inc.,Piscataway, N.J.), a self-complementary oligomer which containstranslational stop signals in all three reading frames, was alsoincluded in the ligation. Upon ligation, the inserted oligomer provideda new translation stop signal for the sCR1 cDNA.

11.2.3. Construction of pBM-CR1c

pBMT3X is a eukaryotic expression vector (Krystal, M., et al., 1986,Proc. Natl. Acad. Sci. USA 83:2709-2713) that contains the humanmetallothionein—1A gene, which confers to cells resistance to increasedlevels of heavy metals such as cadmium. The vector also contains; themouse metallothionein-1 gene that contains an engineered XhoI sitepreceding the initiation codon for the Mt-1 protein. The XhoI site isused as the insertion site for expression of genes under the control ofthe mouse Mt-I promoter.

sCR1c insert (approximately 5.9 kb) was excised from pBSCR1c using XhoIand then ligated to the unique XhoI site of vector pBMT3X. The correctorientation of the sCR1c insert in pBMT3X was determined by restrictiondigestion (Maniatis, T., et al., 1982, Molecular Cloning, A LaboratoryManual, Cold spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Theresulting plasmid was named pBM-CR1c.

11.2.4. Construction of Deletion Mutants pT-CR1c1, pT-CR1c2, pT-CR1c3pT-CR1c4, and pT-CR1c5

Various deletion mutants were also constructed that specifically deletedportions of the sCR1 cDNA (FIG. 20). Each deletion mutant lacked thetransmembrane region of the full length cDNA so that expression of themutants would yield soluble polypeptides.

11.2.4.1. pT-CR1c1

pBSCR1c was digested with SmaI, resulting in two fragments of size 2.56kb and 7.3 kb. These fragments were separated by agarose gelelectrophoresis, and the 7.3 kb fragment was purified and religated toitself. E. coli DH5α cells were made competent (Morrison, D. A., 1979,Meth. Enzymol. 68:326-331) and then transformed with the ligation mix.The resulting plasmid was named pBL-CR1c1. This construct removed 38% ofLHR-B, 100% of LHR-C, and 51% of LHR-D of the CR1c insert. In addition,it regenerated the SmaI site at junction 2335/4894 bp and maintained thecorrect translational frame. pBL-CR1c1 was digested with XhoI and theCR1 insert was separated from the pBluescript® vector. The isolated CR1fragment was then inserted into the unique XhoI site of expressionvector pTCSgpt to produce plasmid pT-CR1c1.

11.2.4.2. PT-CR1c2

pBSCR1c was digested with ClaI and BalI, resulting in two fragments ofsize 3.96 kb and 5.9 kb. These fragments were purified from an agarosegel. Plasmid pBR322 was digested with ClaI and BalI and the 2.9 kbpBR322 fragment was purified and ligated to the 5.9 kb fragment frompBSCR1c. E. coli DH5α cells were transformed with the ligation mix andthe resulting plasmid was termed pBR8.8 This plasmid was digested withXbaI, generating two fragments of size 7.45 kb and 1.35 kb. The 7.45 kbfragment was purified from an agarose gel and religated to itself. Theresulting plasmid, pBR7.45, was digested with ClaI and, BalI, and theisolated 4.5 kb fragment containing the sCR1 cDNA was ligated to the3.96 kb fragment from pBSCR1c, resulting in plasmid pBL-CR1c2. Thisconstruct removed 90% of LHR-B in the sCRI insert, regenerated the XbaIsite at junction 1637/2987 bp, and maintained the correct reading frame.pBL-CR1c2 was digested with XhoI, and the sCR1 insert was separated fromthe pBluescript® vector. The isolated sCR1 fragment was then insertedinto the unique XhoI site of expression vector pTCSgpt to produceplasmid pT-CR1c2.

11.2.4.3. pT-CR1c3

pBSCR1c was digested with NsiI resulting in three fragments of sizes1.09 kb, 1.35 kb, and 7.46 kb. The 7.46 kb fragment was purified from anagarose gel and religated to itself, thus generating plasmid pBL-CR1c3.This construction removed 77% of LHR-A and the rest of the CR1 insert.The NsiI site was regenerated at junction 463/2907 bp. The translationalframe was modified such that a nonsense codon was introduced immediatelyfollowing the regenerated NsiI site. pBL-CR1c3 was digested with XhoIand the sCR1 insert separated from the pBluescript® vector. The isolatedsCR1 fragment was then inserted into the unique XhoI site of expressionvector pTCSgpt to produce plasmid pT-CR1c3.

11.2.4.4. pT-CR1c4

pBSCR1c digested with PstI. The PstI site in the polylinker region ofpBluescript® had been removed during ligation of the CR1 cDNA to thisvector (Example 8.1, supra). The resulting fragments of size 1.35 kb and8.5 kb were separated by gel electrophoresis, and the 8.5 kb fragmentwas purified and religated to itself, generating plasmid pBL-CR1c4. Thisconstruction removed 31% of LHR-A and 69% of LHR-B of the sCR1 insert.The PstI site was regenerated at junction 1074/2424 bp, thus maintainingthe correct reading frame. pBL-CR1c4 was digested with XhoI and the sCR1insert separated from the pBluescript® vector. The isolated sCR1fragment was then inserted into the unique XhoI site of expressionvector pTCSgpt to produce plasmid pT-CR1c4.

11.2.4.5. pT-CR1c5

pBL-CR1c1 was digested with SmaI, thus linearizing the plasmid at theunique SmaI site. The plasmid was dephosphorylated, and ligated tophosphorylated NheI linker containing a Nonsense codon (New EnglandBiolabs, Beverley, Mass.). This type of linker contains a translationalstop codon in all three possible reading frames, and it also contains anNheI restriction site, which faciliates confirming the presence of thenonsense linker in the sCR1 cDNA. The resulting plasmid was namedpBL-CR1c5, and it retained LHR-A and 62% of LHR-B of the sCR1 cDNA.pBL-CR1c5 was digested with XhoI, and the sCR1 insert was separated fromthe pBluescript® vector. The isolated sCR1 fragment was then insertedinto the unique XhoI site of expression vector pTCSgpt to produceplasmid pT-CR1c5.

11.3. Expression of Soluble CR1

As demonstrated herein, the expression of a soluble form of CR1 that canbe secreted from cells in high yield is (t) not limited to one exactsite in the CR1 cDNA to be used for deletion or truncation, and (ii) isalso not limited to the use of a particular expression vector (seeinfra). The ability to produce secreted sCR1 was demonstrated in twodifferent expression systems.

11.3.1. Construction of pTCS Series of Expression Vectors

The pTCS series of expression vectors which were used consists of threeplasmids, each with a unique XhoI cloning site for insertion of cDNAs(FIG. 21). Transcription of the inserted cDNA is driven by a set oftandem promotors. The SV40 early promoter which is located upstream ofthe adenovirus 2 major late promotor (AD2 MLP). Between the beginning ofthe cDNA and the AD2 MLP is the adenovirus tripartite leader.Transcribed mRNAs are terminated at a polyadenylation signal provided bythe murine immunoglobulin kappa (Igκ) sequences located downstream ofthe XhoI cDNA cloning site. Selectable markers xanthine-guaninephosphoribosyltransferase (gpt), dihydrofolate reductase (dhfr), orneomycin resistance (neo^(r)) were provided by the insertion of thecorresponding markers from pSV2gpt, pSV2dhfr, or pSV2neo, respectively.These plasmids were also the source of the bacterial origin ofreplication and beta-lactamase gone for ampicillin resistance. Ingeneral, the choice of which of these vectors to use depends upon whichselectable marker or combination of markers is preferred for selectionof the recombinants.

The complete DNA sequences are known for adenovirus 2 (Ad2), SV40,pSV2cat (Gorman, C., 1985, DNA Cloning, Volume II, A Practical Approach,ed. D. M. Glover, IRL Press, pp. 143-190),and murine immunoglobulinkappa. Sequences are located in the GenBank® database and the NationalBiomedical Research annotation and references. Any of these sequencescould also serve as a source for the appropriate segments of the pTCSvectors.

The vectors pTCSgpt, pTCSneo, and pTCS dhfr were constructed from theintermediate plasmids pEAXgpt and pMLEgpt as follows:

11.3.1.1. Construction of pEAXgpt

Step 1. The Ad2 MLP DNA fragment was derived from M13 mp9/MLP (Concino,M. F., et al., 1983, J. Biol. Chem. 258:8493-8496). This plasmidcontains adenovirus 2 sequences of nucleotides 5778 (XhoI site) to 6231(HindIII site) including the PvuII restriction site at nucleotide 6069and the SacII site at nucleotide 5791 (see NBRF Nucleic database,accession #Gdad2). The XhoI to HindIII fragment had been cloned into theHindIII and SalI sites of M13 mp9 to generate plasmid M13 mp9/MLP.

Plasmid M13 mp9/MLP was digested with EcoRI and HindIII and the smallerMLP containing fragment isolated. A pUC plasmid (Pharmacia, Inc.,Piscataway, N.J.) was also digested with EcoRI and HindIII and thelarger fragment from this plasmid was then ligated to the EcoRI toHindIII MLP fragment. This resulted in a new MLP-containing plasmid withthe, plasmid backbone of pUC. This plasmid was digested with SmaI,ligated to SalI linkers, and recircularized. This new plasmid was thendigested with PvuII which cleaved the plasmid at the PvuII site locatedat position #6069 within the adenovirus 2 insert sequences. Theresulting linear fragment was ligated to XhoI linkers andrecircularized. This plasmid was then digested with XhoI and SalI andthe smaller fragment containing MLP DNA was isolated (fragment #1).

Step 2. Plasmid, pSV2gpt (American Type Culture Collection (ATCC)Accession No. 37145), was digested with PvuII, ligated to SalI linkers,and digested with SalI. The final product was a linear pSV2gpt fragmentthat served as the source of the gpt gene (fragment #2).

Step 3. A murine immunoglobulin Igκ fragment (Hieter, P. A., et al.,1980, Cell 22:197-207) was digested with HaeIII and AvaII and thefragment containing the polyadenylation sequences isolated. In themurine Ig kappa sequence available in the NBRF Nucleic database.(accession #Kcms), the Ig stop codon is at position 1296, followed bythe AvaI. site at 1306, the AATAAA polyadenylation site at. 1484, andthe HaeIII site at 1714. The overhanging ends of this fragment werefilled in with E. coli DNA polymerase, and the fragment was then ligatedto XhoI linkers, and digested with XhoI. This fragment (fragment #3)served as the source of the polyadenylation site.

Step 4. Fragments 1, 2, and 3 were ligated together with T4 DNA ligaseto produce a circular plasmid. The correct orientation of the fragmentsin this plasmid was confirmed by restriction enzyme analysis. Downstreamof the XhoI cDNA cloning site was the murine kappa polyadenylation site,and further downstream from this site was the SV40 promoter and gptgene. Upstream of the XhoI site was the MLP promoter and furtherupstream from this promoter was the bacterial origin of replication andampicillin gene. This plasmid was then digested with SalI and theoverhanging ends filled in with E. coli DNA polymerase. The resultingblunt end fragment was ligated to EcoRI linkers and recircularized withT4 DNA ligase. This final plasmid was designated pEAXgpt.

11.3.1.2. Construction of PMLEgpt

Step 1. Plasmid pMLP CAT (Lee, R. F., et al., 1988, Virology, 165:51-56)is an expression plasmid with a pML vector backbone and contains theadenovirus 2 MLP and tripartite leader sequences 5′ to the CAT gene.pMLP CAT was digested with XhoI and SacII; the XhoI cut at a sitebetween the CAT gene and the L3 region of the tripartite leader, andSacII cut at position #5791 within the adenovirus DNA but 5′ of the MLP.The AD2 MLP and tripartite leader were thus located on this small XhoIto SacII fragment (fragment #4).

Step 2. Plasmid pEAXgpt was digested with XhoI and SacII, and thesmaller MLP containing fragment was discarded. The larger fragment(fragment #5) was isolated. Fragments 4 and 5, both with SacII and XhoIends, were ligated to produce plasmid pMLEgpt.

11.3.1.3. Construction of pTCSgpt

Step 1. pMIEgpt was digested with SacII and the ends filled in with T4DNA polymerase to yield a blunt end fragment (fragment #6). This SacIsite is located at nucleotide 5791 in the Adenovirus 2 sequence, 5′ ofMLP-tripartite leader.

Step 2. pSV2dhfr (ATCC Accession No.37146) wits digested with HindIIIand PvuII. The smaller 342 nucleotide fragment containing the SV40 earlypromoter was blunt ended using the Klenow fragment of E. coli DNApolymerase (fragment #7). Fragments 6 and 7 were ligated with T4 DNAligase. Restriction enzyme analysis confirmed that the fragments werecorrectly oriented to give two, tandem promoters upstream of the XhoIcDNA cloning site, each promoter able to prime RNA synthesis in the samedirection. This plasmid was named pTCSgpt (FIG. 22).

11.3.1.4. Construction of pTCSdhfr

Step 1. pSV2dhfr was digested with HindIII and PvuII, and the largerfragment was then purified from an agarose gel (fragment #8). Thesmaller SV40 early promoter containing fragment was discarded.

Step 2. pTCSgpt was digested with EcoRI and then filled in with theKlenow fragment of E. coli DNA polymerase to generate blunt ends. Thislinear fragment was then digested with HindIII, and the fragment (about1600 nucleotides) containing the PTCS transcription unit of SV40promoter, MLP, tripartite leader, XhoI cDNA cloning site, murine Igλsequences, and second SV40 promoter was isolated (fragment #9). Thisfragment had one flush end and one HindIII overhanging end. Ligation offragments 8 and 9 generated plasmid pTCSdhfr.

11.3.1.5. Construction of pTCSneo

Step 1. pSV2neo (ATCC No. 37149) was digested with HindIII and BamHI,and the larger fragment (fragment #10) was isolated. This fragmentcontained the plasmid backbone and neo gene.

Step 2. pTCSdhfr was digested with HindIII and BamHI, and the PTCStranscription unit (fragment #11) was isolated from an agarose gel afterelectrophoresis of the digestion products. Ligation of fragments 10 and11 generated plasmid pTCSneo.

11.3.2. Expression and Assay of Plasmids pBSCR1c, pBSCR1s and pBM-CR1c,Mammalian Expression Vectors Containing Soluble CR1 Coding Sequences p11.3.2.1. Expression of CR1 Constructs Truncated at Different Positionswithin the CR1 cDNA

Plasmids pBSCR1c and pBSCR1s were constructed (Section 11.1, supra) suchthat most of the cDNA coding regions, except the transmembrane andcytoplasmic regions were preserved (FIG. 20). pBSCR1s is shorter thanpBSCR1c since it is also missing a portion of LHR-D and SCRs 29 and 30that are present in pBSCR1c. The sCR1 portions of these plasmids wereinserted into pTCSgpt, followed by transfection and expression asdescribed infra.

pBSCR1c/pTCSgpt construction: pBSCR1c was digested with XhoI to yieldthe 5.9 kb insert, sCR1c. sCR1c was inserted into the XhoI cDNA cloningsite of pTCSgpt to produce pBSCR1c/pTCSgpt.

pBSCR1s/pTCSgpt construction: pBSCR1s was digested with XhoI and PvuI torelease the sCR1s insert. The ends of the insert were made blunt with T4DNA polymerase. This insert was purified from an agarose gel. VectorpTCSgpt was digested with XhoI, and the overhanging XhoI ends werefilled in with E. coli DNA polymerase I. Next, the sCR1s insert wasligated to the blunt end vector to produce pBSCR1s/pTCSgpt.

Plasmids pBSCR1c/pTCSgpt and pBSCR1s/pTCSgpt were digested with FspI,and the resultant linear DNA's were transfected into Chinese HamsterOvary cells that were mutant in the dhfr gene (CHO DUX B11 cells) viacalcium phosphate coprecipitation with plasmid pSV2dhfr. Transfectantswere selected by their ability to grow in DHFR selection medium. Culturesupernatants of transfectant clones were assayed for secreted sCR1 byELISA. Culture supernatants from fifty pBSCR1c/pTCSgpt recombinants wereassayed and the positive recombinants were taken through theamplification process by culturing them in increasing concentrations ofmethotrexate. In addition, pools of transfectants were prepared byco-culturing eight pBSCR1C/pTCSgpt transfectants together per pool andcarrying them through the same amplification process. Results of theamplification are presented in Table VI.

TABLE VI EXPRESSION OF pBSCR1c/pTCSgpt Secreted Soluble CR1 (μg/ml) 500nM CLONE 0 MTX 20 nM MTX 50 nM MTX 100 nM MTX MTX  2* 0.7 3.4 11 10.9  40.04 0.1  6 0.04  9 0.02 10 0.2 11 0.12 12 0.14 13 0.07 14 0.2 15 0.451.1 7.3 9.0 21 0.07 30 0.27 <0.02 <0.02 35*† 0.82 6.3 8.4 10.9 10.9 400.05 41 0.05 50 0.12 52 0.12 POOL A 0.02 B 0.04 C 0.23 D <0.02 <0.02 E0.27 1.1 F 3.6 5.8 9.1 G 0.27 H 0.04 *clones 2 and 35 were chosen forlarge scale production of sCR1. †Clone 35 was subcloned by limitingdilution, and the production of soluble CR1 was determined for eachsubclone. pBSCR1c/pTCSgpt-clone 35.6 was the highest producer, showing17.7 μg/ml mCR1. MTX: methotrexate

Twelve recombinants from pBSCR1s/pTCSgpt were assayed for production ofsoluble CR1 by ELISA. All twelve candidates showed detectable levels ofsecreted sCR1. The best producers gave levels of sCR1 which werecomparable to those produced by the best pBSCR1c/pTCSgpt transfectants.

pBSCR1c/pTCSgpt and pBSCR1s/pTCSgpt recombinants produced soluble CR1with similar levels of production. This indicated that the ability toproduce a soluble CR1 polypeptide was not dependent upon an exacttruncation point within the CR1 cDNA.

11.3.2.2. Expression of sCR1c in Two Different Expression Systems

The truncated CR1 cDNA insert, sCR1c, was inserted into the expressionvector pTCSgpt and expressed as described above. It was also insertedinto the expression vector pBMT3X as described supra in Section 11.2.3,to yield pBM-CR1c. Both these expression vectors have very strongpromoters. Expression of soluble CR1 was tested in both systems todetermine whether one system would produce better yields of secretedpolypeptide.

C127I mouse cells (ATCC Accession No. CRL 1616, Rockville, Md.) weretransfected with pBM-CR1c using the calcium phosphate method (Graham, F.L. and van der Eb, A. J., 1973, Virology 52:456-467). After glycerolshock, the cells were refed with D-MEM medium containing 10% fetalbovine serum and 2 mM L-glutamine, and incubated at 37° C. for 48 hours.Thereafter, the cells were trypsinized, and split at 1:5 and 1:10 ratiosinto complete D-MEM medium plus 10 μM cadmium chloride.Cadmium-resistant colonies appeared within 10 days. Ten colonies wereremoved with the use of cloning cylinders. Each colony was transferredto a 60 mm petri dish containing complete D-MEM medium, and incubated at37° C., 5% CO₂ until the cells reached confluency. Thereafter, for eachdish, the cells were trypsinized and divided into three 60 am dishes tobe used for preparation of frozen cell stocks, RNA extraction, and ELISAtest of the cell medium for the presence of secreted sCR1c.

When cell medium from each confluent petari dish was removed andsubjected to ELISA analysis, all pBM-CR1c clones tested were positivefor soluble CR1 production. The levels of secreted sCR1 from thepBM-CR1c recombinants were comparable to those from the pBSCR1c/pTCSgptrecombinants. This indicated that the ability to produce high levels ofsecreted sCR1 polypeptide was not dependent upon the use of onlycertain-promoters or expression systems.

11.3.3. Expression and Assay of Plasmids pT-CR1c1, pT-CR1c2, pT-CR1c3,pT-CR1c4, and pT-CR1c5, Mammalian Expression Vectors Containing SolubleCR1 Coding Sequences

The pT-CR1c series of deletion mutants were missing the transmembraneand cytoplasmic domains, as were the constructs, pBSCR1c and pBSCR1s. Inaddition, the deletion mutants also contained fairly large deletions ofvarious LHR regions of the CR1 cDNA (see FIG. 20). The deletion mutantswere expressed in CHO DUX B11 cells and the levels of soluble CR1polypeptide produced were measured.

For each deletion construct, forty different pools of clones wereselected for ELISA analysis to determine whether soluble CR1polypeptides were being produced. All five pT-CR1c constructs were foundto be secreting sCR1 into the cell culture medium, an determined eitherby ELISA or by the presence of functional activity in the call culturemedia. Supernatants from cells transfected with four of the five pT-CR1cconstructs were producing sCR1 that was functional as determined by ahemolytic assay (see Table VII and Section 13.2, infra).

TABLE VII PRODUCTION OF FUNCTIONAL sCR1 FRAGMENTS* Construct ELISAHemolytic Assay pT-CR1c1 − + pT-CR1c2 + + pT-CR1c3 − + pT-CR1c4 + Notdetermined pT-CR1c5 − + *Supernatants tested for ELISA or hemolyticassays were obtained either from cultures growing in T75 flasks or in 24well dishes. Since various amounts of soluble CR1 could have accumulatedin the culture supernatants under these conditions, the results shownare qualitative. (+) indicates the production of functional sCR1 asdetected by the indicated assay.

The fact that the deletion mutants were also able to produce solubleCR1, further demonstrated that the ability to express sCR1 was notdependent upon one exact genetic modification of the CR1 cDNA. As longas the transmembrane regions were deleted, all constructs were able toproduce a soluble polypeptide.

12. EXAMPLE Production and Purification of Soluble CR1

Large quantities of sCR1 were produced in a hollow fiber bioreactorsystem. The quantities of sCR1 obtained were proportional to therelative yield of the inoculated recombinant clones. For optimalpurification results, a serum-free medium was chosen that resulted inhigh production levels of sCR1 in the absence of large quantities ofexogenously added fetal calf serum polypeptides.

12.1. Large Scale Production of Soluble CR1

A Cell-Pharm™ Cell Culture System I (CD Medical, Inc., Miami Lakes,Fla.), equipped with a model IV-L-hollow fiber bioreactor (30 kDmolecular weight cutoff), was assembled under sterile conditions. Twoclones (clone 2 and clone 35 of pBSCR1c/pTCSgpt) were expanded intoeight T-225 flasks. At confluency, the cells were trypsinized, washed,pelleted, and resuspended in culture media. Approximately 5×10⁸ cells ofclone 2 and 10×10⁸ cells of clone 35 were inoculated into two separatehollow fiber bioreactors. A 20 liter feed reservoir of alpha-MEM plus10% fetal calf serum, 8 mM L-glutamine, 100 μg/mlpenicillin-streptomycin and the appropriate concentration ofmethotrexate (50 nN for clone 2; 500 nM for clone 35) was used. Premixedgas (5% CO₂ in air) was bubbled into the reservoir medium through theoxygenator to maintain pH. Media recirculation, replacement and gasflow-rates were adjusted to yield maximum production. Samples wereharvested through inoculating ports, centrifuged at 1000 rpm for 10minutes, filtered through a 0.22 μK pore size filter, and kept at 4° C.before purification. Harvest volume and-frequency were increasedgradually from 25 ml, three times per week at the beginning of theculture, to 40 ml, five times a week after 2-3 months. The production ofsCR1 was assayed by a CR1 ELISA. The yields of clone 2 and clone 35 forthe first month after inoculation were 66 μ/day and 1060 μg/day,respectively. These yields increased as the cultures became established.

12.1.1. Production of sCR1 in Serum-Free Media

Two commercially available serum-free media were tested for theirability to support cell growth and production of sCR1. A confluent T75flask of pBSCR1c/pTCSgpt clone 35 was divided into two T75 flasks. Oneflask was cultured with alpha MEN, supplemented with 10% fetal calfserum, L-glutamine, antibiotics, and 500 nM methotrexate. The otherflask was weaned stepwise from 5%, 1%, 0.5% and no fetal calf serum inalpha MEN plus L-glutamine, antibiotics, 500 nM methotrexate plus HB CHOgrowth supplement (Hana Biologics, Inc., Alameda, Calif.). The cellgrowth and sCR1 production levels of the two flasks were compared. Thegrowth of the cells in the serum-free media never reached confluency.The levels of sCR1 production are given in Table VIII. In each case, thelevel of sCR1 production was best when cells were grown in 10% fetalcalf serum. For comparison, the levels found at day 14 in serum-freemedia were 1.4×10¹⁰ ghosts/ml as compared to 4.2×10¹⁰ ghosts/ml for 10%fetal calf serum supplemented media.

TABLE VIII PRODUCTION OF sCR1 IN SERUM-FREE MEDIA SUPPLEMENTED WITH CHOGROWTH SUPPLEMENT VERSUS 10% FETAL CALF SERUM CONTAINING MEDIA Day 4 Day7 Day 11 Day 14 Flask 1 CHO Growth 5% FCS 1% FCS 0.5% FCS 0% FCSSupplement 2.6 2.4 2.95 1.4 Plus Flask 2 10% FCS 4.8 3.85 4.3 4.2*expressed as 10¹⁰ ghosts/ml

Cell growth and sCR1 production in recombinants were tested using asecond source of serum-free media (CHO-1, Ventrex Laboratories, Inc.,Portland, Me.). Since it was not necessary to wean serum-grown cellsinto this media, cells were thawed and cultured directly in theserum-free media. This media consists of a DME-F12 base and a growthadditive. Equal numbers of cells were thawed and seeded into separatewells in a 24-well plate. After the cells had attached, the media wasdiscarded, and either 10% fetal calf serum containing media orserum-free media was added to appropriate wells. Each condition wasperformed in duplicate. Unlike the previously tested serum-free media,the CHO-1, Ventrex Laboratories media yielded similar levels of callgrowth as did the fetal calf serum containing media.

12.1.2. Conclusions

The above-described results indicated that sCR1 producing CHO cellscould be maintained in a defined serum-free media. This resulted in asavings in the cost of culture media for large scale production runs. Afurther advantage was that purification of sCR1 from the cell culturesupernatants was simplified, since no fetal calf serum proteins had tobe removed.

12.2. Purification of Soluble CR1

With the advent of specific anti-CR1 antibodies, it became possible toreplace the many chromatographic steps needed to produce purified CR1with a simplified two step procedure. This increased the yields of CR1proteins that could be obtained to approximately 1-5 mg CR1 per 5.9×10¹³erythrocytes (Wong, W. W., et al., 1985, J. Immunol. Methods82:303-313). However, since the reported purification was ofmembrane-bound forms of CR1, it was always necessary to solubilize theCR1 containing material in detergents.

Soluble CR1 produced by recombinant transfectants does not have to besolubilized with detergents for purification; it is already soluble.Although soluble CR1 can be purified by anti-CR1 antibody chromatography(see below), this procedure does not lend itself easily to large-scaleproduction. The extent of scale-up is limited by the amount of anti-CR1antibody that can be obtained for preparing the antibody matrix of theantibody purification columns. In addition, the high binding affinity ofan antibody such as YZ-1 for CR1 means that rather harsh conditions, forexample pH 12.2, have to be used to remove the bound sCR1 product fromthe antibody matrix (Wong, W. W.,, et al., 1985, J. Immunol. Methods82:303-313).

To have the capacity of purifying very large quantities of soluble CR1,purification procedures involving HPLC columns were developed. TheseHPLC columns can easily be scaled up to produce even larger quantitiesof purified soluble CR1. In addition, they do not require harshconditions for the elution and recovery of sCR1.

12.2.1 Antibody Affinity Column Purification

12.2.1.1. Methods

For antibody affinity purification of sCR1, 100 mg of monoclonalantibody YZ-1 was covalently coupled to 7 mg of AffiGel-10 (BioRad,Richmond, Calif.) according to the manufacturer's instructions. CR1containing supernatant from cell cultures was incubated with theimmobilized YZ-1 in a flask rocking at 4° C. overnight. The material waspoured into a glass column and washed extensively with 10 mM Hepes, 0.1M NaCl, pH 7. The sCR1 was eluted using 20 mM sodium phosphate, 0.7.MNaCl, pH 12 (Yoon, S. H. and Fearon, D. T., 1985, J. Immunol.134:3332-3338). Eluted fractions were tested for the presence of proteinusing the Biorad Protein Assay (BioRad, Richmond, Calif.). Samplescontaining protein were immediately pooled and dialyzed in 0.1 M HepespH 7 overnight (2×1 liters) at 4° C. The sample was then dialyzed in PBSPresence of sCR1 was analyzed by CR1 ELISA.

12.2.1.2. Results

Cell culture supernatant containing sCR1l produced by transfectantpBSCR1c/pTCSgpt clone 2 was loaded onto the anti-CR1 antibody affinitycolumn and the peak sCR1 fractions pooled. An aliquot of this purifiedmaterial was run on a 4-20% SDS-PAGE gel (DAIICHI; Inc., polyacrylamidegels; modified procedure of Laamli, U.K., 1970, Nature 227:680-685).Under reducing conditions, the apparent molecular weight of soluble CR1was about 224,000 daltons (FIG. 23). This purified CR1 was also shown tobe active by its ability to inhibit complement-mediated hemolysis aswell as C5a and C3 a production (Section 13, infra).

12.2.2. CR1 Purification by HPLC

12.2.2.1. Methods

12.2.2.1.1. Starting Material

When cultures were first established in the bioreactors, the levels ofsCR1 production were lower than when the cultures had been growing forseveral months. Generally there was a period of several weeks before thecells in the bioreactor reached confluency and produced maximum levelsof sCR1. Cell culture supernatants with low levels of sCR1 could beconcentrated before purification by either ammonium sulfateprecipitation or by ultrafiltration. Ammonium sulfate fractionation ofsupernatants over the range of 60 to 80% saturation precipitated SCR1 inessentially equivalent yields. The precipitate was dissolved in aminimum volume and dialyzed into starting buffer for the cation exchangeHPLC. Alternatively, the CHO cell culture supernatants could beconcentrated by ultrafiltration and dialyzed into starting buffer forcation exchange chromatography.

As the bioreactors produced higher concentrations of soluble CR1, theCHO cell culture supernatants from these cultures could be dialyzeddirectly into starting buffer for cation exchange chromatography.

12.2.2.1.2. Cation Exchange HPLC Procedure

Samples were dialyzed into starting buffer (0.02 M sodium phosphate,0.06 N sodium chloride, pH 7.0) and then filtered through a 0. 2 μmfilter to remove any particulate material. The sample was then loadedonto a cation exchange high pressure liquid chromatography column (10cm×10 mm, Hydropore-SCX HPLC column from Rainin). The column was washedand eluted with a sodium chloride gradient developed using 0.02 Mphosphate, 0.5 N NaCl, pH 7.0. The sCR1 eluted somewhere between 0.06 Nand 0.25 N NaCl. Elution was monitored by absorbance at 280 nm and byELISA.

12.2.2.1.3. Anion Exchange HPLC Procedure

If desired, further purification of the cation HPLC purified sCR1 couldbe obtained by anion HPLC. Peak fractions from the cation HPLC weredialyzed into the starting buffer for anion HPLC. Samples were loadedand the column (Hydropore-AX from Rainin) was washed in 0.01 M phosphatepH 7.5. The column was eluted with a series of steps and gradientsdeveloped using 0.01 M phosphate, 0.5 N NaCl, pH 7.5. The sCR1 elutedsomewhere between 0.0 N and 0.3 N NaCl. Elution was monitored as beforefor cation exchange HPLC. The concentrations and pH of the cation andanion HPLC column buffers are given as examples only. Other bufferconcentrations, salt conditions, or pH conditions would also work.

12.2.2.1.4. Western Blot Analysis

Western blotting was performed using a modified procedure from Towbin,H., et al., 1979, Proc. Natl. Acad.

Sci. USA, 76:4350-4354. Briefly, purified sCR1 was run on a 4-20%SDS-PAGE, transferred to nitrocellulose, specifically probed withanti-CR1 (mouse mAb YZ-1 or J3D3), and detected with goat anti-mouseantibody conjugated with alkaline phosphatase.

12.2.2.2. Results

For a typical run, 50-100 ml of supernatant from a bioreactor culturewere dialyzed into starting buffer and loaded onto a 10 cm×10 am cationexchange HPLC. The peak fractions were determined by ELISA andabsorbance at 280 nm, and were pooled. The protein concentration of thepool wars determined by absorbance at 280 nm (ε(1%) at 280 nm=10, asestimated from the CR1c amino acid composition). Several tens ofmilligrams were purified from 100 ml of amplified culture supernatant.

As an example, 100 ml of culture supernatant from transfectantpBSCR1c/pTCSgpt clone 2 produced 22 mg of purified sCR1, as determinedby absorbance at 280 nm, when purified by cation HPLC (FIG. 24). Whenmonitored by CR1 ELISA, the yield was calculated to be 202% with another13% in the flow-through or column wash fraction. The greater than 100%yield probably reflects matrix effects in the ELISA.

Given the rates that culture supernatant can be withdrawn from abioreactor, it should be possible at this level of methotrexateamplification to produce about 100 mg of purified soluble CR1 per weekper bioreactor. Some ways in which this level of production can bescaled up, include amplifying the starting cultures to a maximum extentwith methotrexate prior to seeding th bioreactor, increasing the numberof bioreactors in production at any one time, and using larger capacityHPLC columns.

12.2.2.3. Characterization of Purified Soluble CR1

The sCR1 containing peak fraction from the cation HPLC (FIG. 24) wasfurther purified on an anion HPLC. The purity of the sCR1 material atthe various steps was tested by SDS-PAGE (FIG. 25). The smaller bandsseen in these heavily loaded gels represent fragments of sCR1 asdetermined by Western Blot analysis using anti-CR1 monoclonalantibodies, YZ1 or J3D3. The fragment SCR1 bands were not soon in mostpreparations.

The functional activity of purified sCR1 was tested by its ability toinhibit classical complement-mediated hemolysis by 50% at a purifiedsCR1 concentration of 0.25 μg/ml. The purified soluble CR1 was also ableto inhibit classical complement C5a production by 50% at 5 μg/l and C3aproduction by 50% at 13 μg/ml (see Section 13, infra).

12.2.2.4. Conclusions

As described supra, we developed an improved method for the purificationof soluble CR1 that can easily be scaled up to produce the quantities ofsCR1 needed for therapeutic applications. The basic elements of thisprocedure included a starting material that is already soluble, thuseliminating the requirement of solubilizing membrane bound CR1 withdetergents. The reduction of fetal calf serum concentrations in thebioreactor cultures and/or the use of alternative culture media in thesecultures eliminated the need to remove high concentrations of extraneousproteins from the sCR1-containing starting material during subsequentpurification. Furthermore, the development of an HPLC procedure forpurification provided a method for large-scale purification. Eithercation HPLC or a combination of cation HPLC followed by anion exchangeHPLC can be used for purification. Substantially pure soluble CR1 inhigh yield can be achieved by this procedure in only 1 or 2 steps.

13. EXAMPLE Demonstration of in vitro Activity of Soluble CR1 13.1.Inhibition of the Neutrophil Oxidative Burst

In the reperfusion injury model of tissue damage incurred during amyocardial infarction, activated complement components induce neutrophiladhesion and activation. The activated neutrophil undergoes an oxidativeburst creating highly toxic oxygen radicals. These and other potentialtoxins are released during neutrophil degranulation, damaging thesurrounding tissue. Soluble CR1 may reduce the area of damaged tissue bypreventing the generation of C3a and C5a, the complement componentsinvolved in neutrophil activation.

To monitor the ability of soluble CR1 to block the generation of C5aduring complement activation in vitro, a bioassay which can quantitatethe generation of oxygen radicals produced by neutrophils during a C5ainduced oxygen burst was used (Bass, D.A., et al., 1983, J. Immunol.130:1910-1917). This assay employs dichlorofluorescin diacetate (DCFDA),a lipid soluble molecule which can enter cells, become trapped, and turnhighly fluorescent upon oxidation. 13.1.1. Materials and Methods13.1.1.1. Materials

Fresh whole blood, human complement sources (Beth Israel Hospital,Boston, Mass.), dried Baker's yeast, PBS with 0.1% gelatin and 5 mMglucose, 100 mM EDTA, 10 mM DCFDA in HBSS (Kodak), Red blood cell (RBC)lysing buffer (Ortho Diagnostics), purified C5a (Sigma Chemical Co., St.Louis, MO.), and soluble CR1 were used.

13.1.1.2. Preparation of Neutrophils

Neutrophils were prepared an described by Bass (1983, J. Immunol.130:1910-1917). 2.0 ml of whole blood was washed 3 times inPBS-gelatin-glucose, resuspended in 5 ml of 10 μM DCFDA in HBSS plus 5ml PBS-gelatin-glucose and incubated for 15 minutes at 37° C. Cells werethen centrifuged and resuspended in 2.0 ml PBS-gelatin-glucose plus 5 mMEDTA.

13.1.1.3. Preparation of Yeast Particles

Dried baker's yeast was resuspended in H₂O, washed 2 times and boiledfor 30 minutes. Particles were rewashed 2 times in H₂O and resuspendedat 0.5 grams/ml in H₂O (Simpson, P. J., et al., supra).

13.1.1.4. Activation of Neutrophils by Purified C5a

100 μl of DCFDA-loaded cells were treated with RBC lysing buffer, washedone time in PBS-gelatin-glucose-EDTA and resuspended in 1.0 ml ofPBS-gelatin-glucose. Fifty μl of purified C5a at 200 ng/al or controlwas added to 0.5 ml of target cells at 37° C. and analyzed on the flowcytometer at various time intervals.

13.1.1.5. Activation of Neutrophils by Purified C5a in Human Serum orPlasma

100 μl of DCFDA-loaded cells were incubated with 50 μl of C5a diluted1:1 in human serum or heparinized plasma (100 ng/ml) or control at 37°C. for 30 minutes. The RBC's were lysed out, and the neutrophils wereanalyzed on a flow cytometer.

13.1.1.6. Activation of Neutrophils by Yeast Particle-Activated HumanSerum or Plasma

425 μl of fresh frozen serum and plasma plus 50 μl of sCR1 or controlwere incubated with 25 μl of yeast particles at 37° C. for 30 minutes.The complement-activated and control samples were then centrifuged toremove the yeast particles. The 2-fold dilutions of each of thesesamples were performed in PBS-gelatin-glucose-EDTA. 50 μl of each serialdilution of control and activated serum and plasma was added to 50 μl ofDCFDA-loaded target cells and incubated at 37° C. for 30 minutes. TheRBC's were then lysed out, and neutrophils were analyzed by flowcytometry.

13.1.2. Results

13.1.2.1. C5a Induces an Oxygen Burst in Human Neutrophils which can beMeasured Using DCFDA

FIGS. 26a -26 g show a rapid increase in fluorescence intensity of thehuman neutrophils after stimulation with purified C5a. Within fourminutes after addition of C5a (20 ng/ml final concentration), theneutrophils were 10-fold brighter than control DCFDA-loaded neutrophils.By 20 minutes, the neutrophils were 20-fold as bright as controls. Thisassay seems to be a sensitive indicator of C5a.

13.1.2.2. Human Serum Blocks the Oxygen Burst Effects of Purified C5a onNeutrophils

No increase in fluorescent intensity was observed in neutrophils loadedwith DCFDA and incubated with purified C5a diluted in human serum. Thiseffect may be due to platelet derived growth factor (PDGF) released fromplatelets during clotting. It has been shown that low levels of PDGF caninhibit C5a-induced neutrophil activation (Wilson, E., et al., 1987,Proc. Natl. Acad. Sci. USA 84:2213-2217).

13.1.2.3. Heparinized Plasma does not Block the Effects of C5a onNeutrophils

C5a diluted 1:1 in heparinized plasma induced an oxygen burst in DCFDAloaded neutrophils. Although not as dramatic as C5a in buffer, there wasa ten-fold increase in fluorescent intensity after a 30 minuteincubation with the neutrophils. The decreased signal may be caused byPDGF release during phlebotomy or plasma isolation. More gentle andrapid isolation of the plasma from the cellular components of blood mayminimize the release of PDGF and allow for better C5a function.

13.1.2.4. sCR1 Present During Complement Activation Blocks C5aGeneration

Zymosan induced activation of human complement in the presence ofsoluble CR1 showed reduced C5a activity as measured with the DCFDAassay. As can be seen in FIGS. 27a-27 c the 1:16 dilution of humanplasma activated in the presence of sCR1 generated 70% less fluorescenceintensity increase in the neutrophils as the 1:16 diluted plasmaactivated without sCR1 present. This implies inhibition of C5ageneration by sCR1. Further optimization of the DCFDA assay and plasmacollection should result in a more dynamic and sensitive assay ofsoluble CR1 activity.

13.2. Inhibition of Complement Mediated Hemolysis

13.2.1. Methods

The ability to inhibit complement was tested by assaying for inhibitionof complement-mediated red cell lysis (hemolysis). The inhibition ofhemolysis was determined as a function of soluble CR1 concentration. ThesCR1 samples to be tested were diluted in 0.1 M Hepes buffer (0.15 NNaCl, pH 7.4), and 50 μl were added to each well of a V-bottommicrotiter plate typically in triplicate. Human serum, used as thecomplement source, was diluted 1 to 125 in Hepes buffer, and 50 μl wereadded to each well. Next, commercially available sheep erythrocytes withanti-sheep antibody (Diamedix Cat. No. 789-002) were used as receivedand added 100 μl/well to initiate the complement pathway leading tohemolysis. The plate was incubated for 60 minutes at 37° C. and thencentrifuged at 500×g for 10 minutes. The supernatants were removed andplaced in a flat-bottom microtiter plate. The extent of hemolysis wasmeasured as a function of the sample absorbance at 410 nm. The maximalabsorbance (corresponding to maximal hemolysis), A_(max)′ was obtainedfrom the absorbance of an erythrocyte sample containing only human serumA_(S), minus the absorbance of a sample containing only the red cells,A_(O). Thus, A_(max)=A_(S)−A_(O). The difference between the absorbanceof an erythrocyte sample containing both human serum and sCR1, and theabsorbance of a cell sample containing sCR1 only, was defined asA_(sample). The inhibition, IH, was expressed as the fraction(A_(max)−A_(sample)/A_(max)) and IH₅₀ was defined as the concentrationof sCR1 required to produce a value of IH=½. To monitor chromatographyfractions, the serum-free controls were not included and anti-complementactivity was monitored qualitatively as a decrease in the absorbance at410 nm of the sample.

The hemolytic assay described above was also used to assess thecapability of human recombinant sCR1 to inhibit sheep red cell lysis bycomplement from other species, such as guinea pig and rat. For eachspecies, fresh-frozen serum or freshly lyophilized serum or plasma wasused as a complement source. In some cases sera were obtainedcommercially (Sigma Chemical Company, St. Louis, Mo.).

The serum was first titered for its capacity to lyse activated redcells. The greatest dilution which yielded at least 80% maximal red celllysis was chosen to assess the effects of added human sCR1. The assaywas then performed as described above, substituting animal for humanserum at the preferred dilution.

13.2.2. Results

As indicated in FIG. 28, purified sCR1 inhibited classicalcomplement-mediated lysis by 50% at a sCR1 concentration of 0.12 μg/ml.The ability of antibody affinity purified sCR1 to inhibit the hemolyticassay was compared to that of unpurified material (sCR1 containing cellculture supernatant). The purified sCR1 had activity comparable to thatof the unpurified sCR1, with both producing 50% inhibition in thehemolytic assay at 1.6×10⁸ ghosts/ml. This indicated that thepurification procedure was not substantially diminishing the functionalactivity of the final sCR1 product.

To determine if purified sCR1 could be stored frozen, an aliquot wasstored at −70° C. for one week. The concentration of the frozen sCR1 wasthe same as the nonfrozen sCR1, as determined by absorbance at 280 nmand CR1 ELISA. The frozen sCR1 also had the same activity as thenonfrozen sCR1 as determined by inhibition of hemolysis.

The ability of human recombinant sCR1 to inhibit hemolysis mediated bycomplement from several species is summarized in Table IX.

TABLE IX HEMOLYSIS OF SENSITIZED SHEEP RBC USING COMPLEMENT FROM VARIOUSANIMAL SERA Inhibition Final Conc. Inhibition (IH)** IH₅₀** Animal SerumUsed by sCRI (ghost/ml) (ghost/ml) guinea 1:500 Yes 66%(2.6 × 10⁹) 1.0 ×10⁹ pig* human 1:500 Yes 94%(2.5 × 10⁹) 2.0 × 10⁸ human 1.312 Yes94%(1.2 × 10⁹) 1.0 × 10⁷ rat 1:200 Yes 85%(2.6 × 10⁹) 2.4 × 10⁸ rat*1:200 Yes 77%(3.8 × 10⁹) 1.0 × 10⁹ dog 1:50 No rabbit* 1:20 No mouse*1:5 No *lyophilized sera obtained commercially (Sigma Chemical Co., St.Louis, Mo. **as defined in text (Section 13.2)

Both guinea pig and rat complement appeared to be inhibited by humansCR1. The lack of clear inhibition for other species may reflect (a) theinappropriateness of using rabbit antibodies and sheep erythrocytes inthe assay system, or (b) the high concentration of serum required forhemolysis in this system.

13.3. Inhibition of C3a and C5a Production

13.3.1. Methods

The ability to inhibit complement was also tested by assaying forspecific inhibition of C3a and C5a production. For all experiments, asingle human serum pool, to be used as a source of complement, wasaliquoted and stored frozen at −70° C. Human IgG was heat-aggregated,aliquoted, and stored frozen at −70° C. For each experiment, serumaliquots were equilibrated at 37° C. with varying concentrations of sCR1to be tested. The complement pathway was initiated by the addition ofaggregated human IgG. Control samples containing no IgG were alwaysincluded. After a fixed reaction time of 15 minutes (determined in anearlier time-course study to provide a convenient time interval duringwhich the production of C5a or C3a is nearly complete, i.e., greaterthan 90%), the levels of the released complement peptides (C5a or C3a)were determined by radioimmunoassay using commercially availableradioimmunoassay (RIA) kits (C5a RIA, Amersham Cat No. RPA.520; C3a RIA,Amersham Cat. No. RPA.518) in modified procedures.

Since a competitive immunoassay was used, complement peptide (C5a andC3a) concentrations varied inversely with the counts. The counts bound(CB) for a sample were defined as the total counts (in counts perminute, cpm) measured in the pellet.

The y-axis in FIG. 29 represents the fraction inhibition. The fractioninhibition is equal to the counts bound (CB) for a “sample”, less the CBin the “sample with no sCR1”, divided by the CB for the “no IgG control”less the CB in the “sample with no sCR1.”${INHIBITION} = \frac{\lbrack {( {{CB}\quad {sample}} ) - ( {{CB}\quad {no}\quad {sCR1}} )} \rbrack}{\lbrack {( {{CB}\quad {no}\quad {IgG}} ) - ( {{CB}\quad {no}\quad {sCR1}} )} \rbrack}$

13.3.2. Results

The activity of purified sCR1 was assayed by testing its ability toinhibit C5a and C3a production in an activated human serum sample.

As indicated by FIG. 29, under the conditions tested, purified sCR1 wasable to maximally inhibit C5a production by 100% and C3a by 60%.Inhibition of 50% was observed at sCR1 concentrations of 5 μ/ml for C5aproduction and 15-20 μg/ml for C3a production. The data suggest thatrecombinant sCR1 inhibits the C5 convertase more efficiently than the C3convertase.

14. EXAMPLE: Demonstration of Functional in Vivo Therapeutic Activity ofSoluble CR1 14.1. Soluble CR1 Demonstrates in vivo Function in aReversed Passive Arthus Reaction

The Arthus reaction is a classic immunologically induced inflammatoryresponse caused by injecting antigen locally that then reacts withantibodies in circulation. The major biological response ischaracterized by immune complex deposition, complement fixation,polymorphonuclear (PMN) leukocyte infiltration, release of lysosomalenzymes, vasoactive amine, and local tissue damage (Uriuhura, T. andMovat, H. Z., 1966, Exp. Mol. Pathol. 5:539-558; Cochrane, C. G., 1968,Adv. Immunol. 9:97-162). A modification of the direct Arthus reaction,the reversed passive Arthus reaction (RPAR), has been used as a modelfor identifying anti-inflammatory agents (Pflum, L. R. and Graeme, M.L., 1979, Agents and Actions 9:184-189). In a RPAR, antibody is injectedlocally and antigen is present in the circulation.

When tested in a rat RPAR model, soluble CR1s were able to block thelocal inflammatory reaction. The mechanism of the action of this solubleCR1 function in vivo may be mediated through the inhibition ofcomplement pathway enzymes.

14.1.1. Materials and Methods

Female five week old Sprague Dawley rats (CD strain) weighing about100-125 grams (Charles River Laboratories, Wilmington, Mass.) wereanesthesized with an intraperitoneal injection of 0.1 to 0.3 ml Avertinsolution. This solution was a 1:2 dilution of a stock solution made with1 g tribromoethanol in 15 ml Amel ethanol. The fur on the backs of theanimals was shaved. Next, the tail was warmed, first with warm water andthen with a heat lamp. Using a 1 ml syringe, 0.35 ml of ovalbumin(Calbiochem Corp., San Diego, Calif.) at 5 mg/ml in 0.15 M phosphatebuffered saline (PBS) was injected intravenously into the tail vein,about 1-2 inches from the tip of the tail. Five minutes later, the ratswere injected intradermally with 0.08 ml of 20 mg/ml rabbit Ig fractionof anti-ovalbumin antibody having an antibody titer of 4 mg/ml (OrganonTeknika Corp., Cappel Division, West Chester, Pa.) or with 0.08 ml of 20mg/ml rabbit IgG (Sigma Chemical Co., St. Louis, Mo.), or with PBS. Eachinjection was performed in duplicate and the areas around the injectionwere circled with a marker pen. The rats were then monitored at 1, 4,and 18 hours. After 24 hours, the rats were killed by submerging them indry ice for 3 minutes. Skin samples were dissected from the injectedsites. One of the duplicate samples was fixed in 10% formalin forparaffin embedding and the other frozen for cryostat sections. Tissuesections were prepared and stained with hematoxylin and eosin.

14.1.2. Results

A weak RPAR reaction (e.g., edema and erythema) began to be visibleafter 3 to 5 hours following intradermal injection of anti-ovalbuminantibody. The intensity of the reaction gradually increased until thesize of the reaction reached 3-5 mm in diameter after 24 hours (FIG.30b). No reactions were observed in the rat skin where only non-immunerabbit IgG or PBS was injected.

Under microscopic examination of the tissue sections prepared from thesite of the lesion, many acute inflammatory cells were visible in thedermis, particularly around the blood vessels (FIG. 31b). This istypically recognized as vasculitis and perivasculitis. The tissueindicated a typical inflammatory condition with extensive infiltrationof PMN outside of the blood vessels, the presence of erythrocytes in theconnective tissue, and the loosening of collagen fibers.

14.1.3. Effect of Intradermal Administration of Soluble CR1

A mixture of purified sCR1 was prepared by combining 40 μl of 0.75 mg/mlsCR1 with an equal volume of anti-ovalbumin or normal rabbit IgG or PBS.Either the sCR1: anti-ovalbumin mixture or the sCR1:rabbit IgG mixture,or the sCR1:PBS mixture was injected intradermally into intravenouslyovalbumin primed rats. Barely visible lesions developed in the injectionsites that received sCR1 plus anti-ovalbumin antibody (FIG. 30a). Asexpected, no lesions, developed in the injection sites that receivedsCR1:rabbit IgG or sCR1:PBS. When sections of tissue surrounding the.sCR1:anti-ovalbumin injection sites were examined micro-scopically,clusters of PMN and mononuclear cells could be found surrounding thevenules, but there was no extensive infiltration of PMN or extravasationof erythrocytes (FIG. 31 a). These data indicate that soluble CR1administration caused an inhibition of damage to the endothelial cellsand an inhibition of the inflammatory reaction.

In order to determine the minimum effective dosage of sCR1 that isrequired to block a RPAR in the above ovalbumin rat model, ten-foldserial dilutions (neat, {fraction (1/10)}, {fraction (1/100)}, {fraction(1/1,000)} and {fraction (1/10,000)}) of the 0.75 mg/ml sCR1 stock weretested. Each sCR1 dilution was mixed with an equal volume of neat orone-half dilution of anti-ovalbumin antibody. Each site was injectedwith a total of 80 μl. The ability of sCR1 to inhibit RPAR was dosedependent, with effective reduction of edema observed at 300 ng per site(Table X).

TABLE X EFFECT OF DOSAGE ON THE INHIBITION OF RPAR BY sCR1 sCR1(μg/site) Extent of Remaining RPAR 30 +/− 3 +/− 0.3 +/− 0.03 ++ 0.003++++ 0 ++++

14.2. Pharmacokinetics of in vivo Administered sCR1

The biological half-life of sCR1 in vivo was determined as follows. Ratsof similar age (6 weeks) and body weight (110-125 g) were injectedintravenously with 250 μg of sCR1 in 0.35 ml. At 2 minutes, 5 minutes,10 minutes, 60 minutes, and 24 hours post-injection, the rats weresacrificed and blood was obtained from vena cava puncture. 1-2 ml ofsera from each rat was obtained by centrifugation at 1800 rpm for 10minutes, and the amount of sCR1 in each sample was determined by CR1ELISA. Two-fold dilutions of 1 μg/ml of purified sCR1 spiked intocontrol rat serum or detergent lysates of hemoglobin-free red blood cellghosts (1.6×10⁸ ghosts/ml) were used as CR1 standards. The results areshown in Table XI.

TABLE XI PHARMACOKINETIC DATA ON SERUM CONCENTRATIONS OF INJECTED sCR1WITH TIME Time After Intravenous sCR1 Concentration Injection (μg/ml)Control 0.01  2 min 0.17  5 min 0.80 10 min 1.01 60 min 0.38 24 hrs 0.49

These data indicate that sCR1 can be detected 24 hours followingintravenous injection. At 24 hours, the level of sCR1 in the serum was50% of the peak level that was observed 10 minutes post-injection.

14.3. sCR1 Reduces Infarct Size in Rats with Reperfused InfarctedMyocardium

As described herein, sCR1 which was able to inhibit the activity of thecomplement pathway C3C5 convertase in vitro was also able to reduce theextent of reperfusion injury in an in vivo rat myocardial infarct model.

Myocardial infarction can be induced in a rat by coronary ligation. Ifestablished within the first few hours after myocardial infarction,reperfusion has been shown to reduce the infarct size, to improve theleft ventricular function, and to reduce mortality (Braunwald, E. andKloner, R. A., 1985, J. Clin. Invest. 76:1713-1719). However,reperfusion to a myocardium that is severely ischemic but notirreversibly injured, can itself produce and extend injury. Themechanisms responsible for the reperfusion-induced injury may includeinjury mediated by oxygen free radicals and cellular calcium overload.Leukocytes acting either alone or in concert with microvascularendothelial cells may contribute to this injury. Complement activationmay be involved in this process (Rossen, R. D., et al., 1985, Cir. Res.57:119-130; Crawford, M. H., et al., 1988, Circulation 78:1449-1458).

14.3.1. Methods

14.3.1.1. Induction of Rat Myocardial Infarct

Rats (n=14) weighing between 200 and 250 grams were anesthetized byinhalation of methoxyflurane, and had a right jugular vein cut-down andcannulation. Half (n=7) received 2 ml (1 mg) of sCR1 through thecannula, and half received 2 ml of saline placebo prepared andadministered similarly. The animals had the jugular cannula removed, thejugular vein tied, and the site closed. A left thoracotomy was thenperformed in the fifth to sixth intercostal space, while intermittentpositive pressure ventilation with 95% oxygen and 5% carbon dioxide wasadministered. A pericardiotomy was then performed, and the left coronaryartery occluded by suture ligation within 2-3 mm left of the proximalaorta. The effect of this coronary ligation was to produce a largeregion at risk for anterior transmural infarction. The chest wastransiently closed, while the rats remained under anesthesia. 35 minutesafter occlusion, the chest was reopened and the ligature released. Thistime span was chosen so that a significant proportion of the risk regionwas potentially salvable. The thoracotomy was then permanently closed,and the animals allowed to awaken from anesthesia, usually within 5 to10 minutes post-operatively. 100,000 units of benzathine penicillin Gand 0.25 mg/kg morphine sulfate were administered intramuscularly. Theanimals were maintained on water and standard rat chow for one week andthen sacrificed following heparinization and methoxyflurane anesthesiaby excision of the heart.

14.3.1.2. Morphologic Analysis of Experimental Infarcts: Preparation ofHearts for Study

Following excision of the heart, the aortas were rapidly cannulated andthe coronary arteries perfused first with Krebs Henseleit solution toclear the hearts of blood and clots, and then with 30 mM KCl fordiastolic cardiac arrest. The hearts were fixed by intracoronaryperfusion and immersion in 10% buffered formalin. To adequately controlfilling pressure, the hearts were vented through the mitral value withplastic tubing. After fixation, the hearts were sliced transverselyparallel to the atrioventricular groove in 2 mm sections from base toapex and histologic sections prepared.

14.3.2. Results

Survival was the same in both groups, viz., {fraction (6/7)} ratssurvived 7 days and were analyzed. On gross inspection of the histologicslides, 5 of 6 placebo-treated rats had large transmural myocardialinfarctions (estimated to be al: least 15% of the total left ventricularmass). Only ⅙ surviving sCR1-treated animals had this finding of a largetransmural infarction. The other sCR1-treated animals had small patchyinfarcts comprising much less of the left ventricular mass (less than15%). In fact, most of these were detectable only by microscopy, whereasthe infarcts from placebo-treated rats were apparent by grossinspection.

14.3.3. Conclusions

The results indicate that sCR1 treatment is effective in reducingreperfusion injury in vivo and in ameliorating the effects of myocardialinfarction. To the extent that reperfusion injury can be ameliorated,the absolute amount of salvaged myocardium can be increased and the timewindow for which reperfusion is clinically useful can be extended.Treatment with sCR1 should be a useful concomitant therapy withthrombolytics or balloon coronary angioplasty during acute infarction.

15. Deposit of Microorganisms

E. coli strain DK1/P3 carrying plasmid piABCD (designated PCR1-piABCD),encoding the full-length CR1 protein, was deposited with theAgricultural Research Culture Collection (NRRL), Peoria, Ill., on Mar.31, 1988 and was assigned accession number B-18355. A supplementaldeposit of E coli strain DK1/P3 carrying plasmid pCR1-piABCD wasdeposited under the Budapest Treaty with the Agricultural ResearchPatent Culture Collection (NRRL), Peoria, Ill., on Jul. 17, 1997 andassigned the NRRL accession number B-18355N.

Chinese hamster ovary cell line DUX B11 carrying plasmid pBSCR1c/pTCSgptclone 35.6, encoding a soluble CR1 molecule, was deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Mar. 23, 1989 and was assigned accessionnumber CRL 10052.

The present invention is not to be limited in scope by themicroorganisms deposited since the deposited embodiments are intended assingle illustration of one aspect of the invention and anymicroorganisms which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

It is also understood that all base pair sizes given for nucleotides areapproximate and are used for the purpose of description.

Various references are cited herein, the disclosures of which areincorporated by reference in their entirety.

What is claimed is:
 1. A method for purifying a recombinant, solublecomplement receptor type 1 molecule comprising: (a) expressing acomplement receptor type 1 molecule having an amino acid sequencecomprising the amino acid sequence as depicted in FIG. 1(A) -1(P) fromamino acid 42 to amino acid 1972 and substantially lacking atransmembrane domain in recombinant cell culture such that the moleculeis secreted into the culture medium; (b) subjecting a sample of the cellculture medium to cation exchange high pressure liquid chromatography;and (c) eluting the molecule from the high pressure liquidchromatography column.
 2. The method according to claim 1 which furthercomprises, after step (c): (d) subjecting the molecule eluted accordingto step (c) to anion-exchange high pressure liquid chromatography; and(e) eluting the molecule from the high pressure liquid chromatographycolumn of step (d).
 3. A method for purifying a soluble fragment ofcomplement receptor type 1 (CR1) wherein said CR1 comprises the aminoacid sequence as depicted in FIGS. 1A -1P and wherein said fragment isselected from the group consisting of LHR-A, LHR-B, LHR-C, and LHR-Dsaid method comprising: (a) culturing a host cell transformed with anisolated DNA molecule encoding said fragment such that the fragment isexpressed by the host cell and secreted into the cell culture medium,(b) subjecting a sample of said culture medium to cation exchange highpressure liquid chromatography, and (c) eluting said fragment from thehigh pressure liquid chromatography column.
 4. A method for purifying asoluble fragment of complement receptor type 1 (CR1) wherein said CR1comprises the amino acid sequence as depicted in FIGS. 1A-1P and whereinsaid fragment comprises short consensus repeats (SCRs) selected from thegroup consisting of i. SCRs 1 and 2; ii. SCRs 1,2,3,and4; iii. SCRs 1,2, 3, 4, 5, 6, 7; iv. SCRs 6, 7, 8, 9, 10, 11, and 12; V. SCRs 8 and 9;vi. SCRs 8, 9, 10, and 11; vii. SCRs 8, 9, 10,11, 12, 13, and 14; viii.SCRs 15 and 16; ix. SCRs 12, 13, 14, 15, 16, and 17; x. S CRs 15, 16,17, 18, and 19; xi. SCRs 1 through 17; xii. SCRs 1 through 23; xiii.SCRs 1 through 28; and xiv. SCRs 1 through 30; said method comprising:(a) culturing a host cell transformed with an isolated DNA moleculeencoding said fragment such that the fragment is expressed by the hostcell and secreted into the cell culture medium, (b) subjecting a sampleof said culture medium to cation exchange high pressure liquidchromatography, and (c) eluting said fragment from the high pressureliquid chromatography column.
 5. A method for purifying a solublefragment of complement receptor type 1 (CR1) wherein said CR1 comprisesthe amino acid sequence as depicted in FIGS. 1A-1P and wherein saidfragment is selected from the group consisting of LHR-A, LHR-B, LHR-C,and LHR-D said method comprising: (a) culturing a host cell transformedwith an isolated DNA molecule encoding said fragment such that thefragment is expressed by the host cell and secreted into the cellculture medium, (b) subjecting a sample of said culture medium to cationexchange chromatography, and (c) eluting said fragment from the cationexchange chromatography matrix.
 6. A method for purifying a solublefragment of complement receptor type 1 (CR1) wherein said CR1 comprisesthe amino acid sequence as depicted in FIGS. 1A-1P and wherein saidfragment comprises short consensus repeats (SCRs) selected from thegroup consisting of i. SCRs 1 and 2; ii. SCRs 1, 2, 3, and 4; iii. SCRs1, 2, 3, 4, 5, 6, 7; iv. SCRs 6, 7, 8, 9, 10, 11, and 12; V. SCRs8 and9; vi. SCRs 8, 9, 10, and 11; vii. SCRs 8, 9, 10, 11, 12, 13, and 14;viii. SCRs 15 and 16; ix. SCRs 12, 13, 14, 15, 16, and 17; x. SCRs15,16,17,18, and 19; xi. SCRs 1 through 17; xii. SCRs 1 through 23;xiii. SCRs 1 through 28; and xiv. SCRs 1 through 30; said methodcomprising: (a) culturing a host cell transformed with an isolated DNAmolecule encoding said fragment such that the fragment is expressed bythe host cell and secreted into the cell culture medium, (b) subjectinga sample of said culture medium to cation exchange chromatography, and(c) eluting said fragment from the cation exchange chromatographymatrix.
 7. The method according to claim 3 or claim 4, which furthercomprises, after step (c): (d) subjecting the fragment eluted accordingto step (c) to anion-exchange high pressure liquid chromatography; and(e) eluting said fragment from the anion exchange high pressure liquidchromatography column.
 8. The method according to claim 5 or claim 6,which further comprises, after step (c): (d) subjecting the fragmenteluted according to step (c) to anion exchange chromatography; and (e)eluting the fragment from the anion exchange chromatography matrix. 9.The method according to any one of claims 3, 4, 5, or 6, wherein theculture medium is subjected to a purification step prior to step (b).10. The method according to claim 9, wherein the purification step priorto step (b) is selected from the group consisting of centrifugation,ultrafiltration, precipitation and dialysis.
 11. The method according toclaim 6, wherein said DNA molecule encodes a CR1 protein fragment havingan amino acid sequence comprising the amino acid sequence as depicted inFIG. 1(A) -1(P) from amino acid 42 to amino acid 1972 and substantiallylacking a transmembrane domain.
 12. The method according to claim 8,wherein said DNA molecule encodes a CR1 protein fragment having an aminoacid sequence comprising the amino acid sequence as depicted in FIG.1(A) -1(P) from amino acid 42 to amino acid 1972 and substantiallylacking a transmembrane domain.
 13. The method according to claim 11,wherein the culture medium is subjected to a purification step prior tostep (b) selected from the group consisting of centrifugation,ultrafiltration, precipitation and dialysis.
 14. The method according toclaim 12, wherein the culture medium is subjected to a purification stepprior to step (b) selected from the group consisting of centrifugation,ultrafiltration, precipitation and dialysis.